Dlin-kc2-dma lipid nanoparticle delivery of modified polynucleotides

ABSTRACT

The present disclosure provides, inter alia, formulation compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor. The formulation composition may further include a modified nucleic acid molecule and a delivery agent. The present invention further provides nucleic acids useful for encoding polypeptides capable of modulating a cell&#39;s function and/or activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/714,458, filed Dec. 14, 2012, entitled Modified Nucleoside,Nucleotide, and Nucleic Acid Compositions which claims priority to U.S.Provisional Patent Application No. 61/576,705, filed Dec. 16, 2011,entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions,U.S. Provisional Patent Application No. 61/618,957, filed Apr. 2, 2012,entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions,U.S. Provisional Patent Application No. 61/648,244, filed May 17, 2012,entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions,U.S. Provisional Patent Application No. 61/681,712, filed Aug. 10, 2012,entitled Modified Nucleoside, Nucleotide, Nucleic Acid Compositions andU.S. Provisional Patent Application No. 61/696,381 filed Sep. 4, 2012,entitled Modified Nucleoside, Nucleotide and Nucleic Acid Compositions,and Nucleic Acid Compositions, U.S. Provisional Patent Application No.61/709,303 filed Oct. 3, 2012, entitled Modified Nucleoside, Nucleotideand Nucleic Acid Compositions, U.S. Provisional Patent Application No.61/712,490 filed Oct. 11, 2012, entitled Modified Nucleoside, Nucleotideand Nucleic Acid Compositions and International Application No.PCT/US2012/058519 filed Oct. 3, 2012 Modified Nucleosides, Nucleotides,and Nucleic Acids, And Uses Thereof, the contents of which areincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file, entitledM11CON10SQLST.txt, was created on May 17, 2013 and is 25,673 bytes insize. The information in electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

In general, exogenous unmodified nucleic acid molecules, particularlyviral nucleic acids, introduced into the cell induce an innate immuneresponse which results in cytokine and interferon (IFN) production andultimately cell death. It is of great interest for therapeutics,diagnostics, reagents and for biological assays to be able to deliver anucleic acid, e.g., a ribonucleic acid (RNA), into a cell, such as tocause intracellular translation of the nucleic acid and production ofthe encoded protein instead of generating an innate immune response.Thus, there is a need to develop formulation compositions comprising adelivery agent that can effectively facilitate the in vivo delivery ofnucleic acids to targeted cells without generating an innate immuneresponse.

SUMMARY OF THE INVENTION

The present disclosure provides, inter alia, formulation compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation compositions may further includea modified nucleic acid molecule and a delivery agent. The presentinvention further provides nucleic acids useful for encodingpolypeptides capable of modulating a cell's function and/or activity.

In one aspect a method of producing a polypeptide of interest in amammalian cell or tissue is described. The method comprises contactingthe mammalian cell or tissue with a formulation comprising a modifiedmRNA encoding a polypeptide of interest. The formulation may be, but isnot limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA)microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates(including simple sugars), cationic lipids, fibrin gel, fibrin hydrogel,fibrin glue, fibrin sealant, fibrinogen, thrombin, rapidly eliminatedlipid nanoparticles (reLNPs) and combinations thereof. The modified mRNAmay comprise a purified IVT transcript.

In one embodiment, the formulation comprising the modified mRNA is ananoparticle which may comprise at least one lipid. The lipid may beselected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG andPEGylated lipids. In another aspect, the lipid may be a cationic lipidsuch as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA,DLin-KC2-DMA and DODMA.

The lipid to modified mRNA ration in the formulation may be between 10:1and 30:10. The mean size of the nanoparticle formulation may comprisethe modified mRNA between 60 and 225 nm. The PDI of the nanoparticleformulation comprising the modified mRNA is between 0.03 and 0.15. Thezeta potential of the lipid may be from −10 to +10 at a pH of 7.4

The formulations of modified mRNA may comprise a fusogenic lipid,cholesterol and a PEG lipid. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC.

The mammalian cell or tissue may be contacted using a device such as,but not limited to, a syringe pump, internal osmotic pump and externalosmotic pump.

The formulation of modified mRNA may be a PLGA microsphere which may bebetween 4 and 20 μm in size. The modified mRNA may be released from theformulation at less than 50% in a 48 hour time period. The PLGAmicrosphere formulation may be stable in serum. Stability may bedetermined relative to unformulated modified mRNA in 90%.

The loading weight percent of the modified mRNA PLGA microsphere may beat least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least0.4% or at least 0.5%. The encapsulation efficiency of the modified mRNAin the PLGA microsphere may be at least 50%, at least 70%, at least 90%or at least 97%.

A lipid nanoparticle of the present invention may be formulated in asealant such as, but not limited to, a fibrin sealant.

The mammalian cells or tissues may be contacted by a route ofadministration such as, but not limited to, intravenous, intramuscular,intravitreal, intrathecal, intratumoral, pulmonary and subcutaneous. Themammalian cells or tissues may be contacted using a split dosingschedule. The mammalian cell or tissue may be contacted by injection.The injection may be made to tissue selected from the group consistingof intradermal space, epidermis, subcutaneous tissue and muscle. Thepolypeptide of interest may be produced in the cell or tissue in alocation systemic from the location of contacting.

The polypeptide of interest may be detectable in serum for up to 72hours after contacting. The level of the polypeptide of interest can behigher than the levels prior to dosing. The level of the polypeptide ofinterest may be greater in the serum of female subjects than in theserum of male subjects.

The formulation of modified mRNA may comprise more than one modifiedmRNA. The formulation may have two or three modified mRNA.

The formulation comprising the modified mRNA may comprise a rapidlyeliminated lipid nanoparticle (reLNP) which may comprise a reLNP lipid,fusogenic lipid, cholesterol and a PEG lipid at a molar ratio of50:10:38.5:1.5 (reLNP lipid:fusogenic lipid:cholesterol:PEG lipid). Thefusogenic lipid may be DSPC and the PEG lipid may be PEG-c-DOMG. ThereLNP lipid may be DLin-DMA with an internal or terminal ester orDLin-MC3-DMA with an internal or terminal ester. The total lipid tomodified mRNA weight ration may be between 10:1 and 30:1.

The formulation comprising modified mRNA may comprise a fibrin sealant.

The formulation comprising modified mRNA may comprise a lipidoid wherethe lipid is selected from the group consisting of C12-200 and 98N12-5.

The formulation comprising modified mRNA may include a polymer. Thepolymer may be coated, covered, surrounded, enclosed or comprise a layerof a hydrogel or surgical sealant. The polymer may be selected from thegroup consisting of PLGA, ethylene vinyl acetate, poloxamer andGELSITE®.

A polypeptide of interest may be produced in a mammalian cell or tissueby contacting the mammalian cell or tissue with a buffer formulationcomprising a modified mRNA encoding the polypeptide of interest. Thebuffer formulation may be selected from, but is not limited to, slaine,phosphate buffered saline and Ringer's lactate. The buffer formulationmay comprise a calcium concentration of between 1 to 10 mM. The modifiedmRNA in the buffer formulation may comprise a purified IVT transcript.

A pharmacologic effect in a primate may be produced by contacting theprimate with a composition comprising a formulated modified mRNAencoding a polypeptide of interest. The modified mRNA may comprise apurified IVT transcript and/or may be formulated in nanoparticles,poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex,liposome, polymers, carbohydrates (including simple sugars), cationiclipids, fibrin gel, fibrin hydrogel, fibrin glue, fibrin sealant,fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNPs)and combinations thereof. The pharmacological effect may be greater thanthe pharmacologic effect associated with a therapeutic agent and/orcomposition known to produce said pharmacologic effect. The compositionmay comprise a formulated or unformulated modified mRNA. Thepharmacologic effect may result in a therapeutically effective outcomeof a disease, disorder, condition or infection. Such therapeuticallyeffective outcome may include, but is not limited to, treatment,improvement of one or more symptoms, diagnosis, prevention, and delay ofonset. The pharmacologic effect may include, but is not limited to,change in cell count, alteration in serum chemistry, alteration ofenzyme activity, increase in hemoglobin, and increase in hematocrit.

In one embodiment, the present disclosure provides a formulationcomposition which comprises a modified nucleic acid molecule and adelivery agent. The modified nucleic acid molecule may be selected fromthe group consisting of DNA, complimentary DNA (cDNA), RNA, messengerRNA (mRNA), RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA,antisense RNA, ribozymes, catalytic DNA, RNA that induce triple helixformation, aptamers, vectors and combinations thereof. If the modifiednucleic acid molecule is mRNA the mRNA may be derived from cDNA.

In one embodiment, the modified nucleic acid molecule may comprise atleast one modification and a translatable region. In some instances, themodified nucleic acid comprises at least two modifications and atranslatable region. The modification may be located on the backboneand/or a nucleoside of the nucleic acid molecule. The modification maybe located on both a nucleoside and a backbone linkage.

In one embodiment, a modification may be located on the backbone linkageof the modified nucleic acid molecule. The backbone linkage may bemodified by replacing of one or more oxygen atoms. The modification ofthe backbone linkage may comprise replacing at least one phosphodiesterlinkage with a phosphorothioate linkage.

In one embodiment, a modification may be located on a nucleoside of themodified nucleic acid molecule. The modification on the nucleoside maybe located on the sugar of said nucleoside. The modification of thenucleoside may occur at the 2′ position on the nucleoside.

The nucleoside modification may include a compound selected from thegroup consisting of pyridin-4-one ribonucleoside, 5-aza-uridine,2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. Inanother embodiment, the modifications are independently selected fromthe group consisting of 5-methylcytosine, pseudouridine and1-methylpseudouridine

In one embodiment, a modification may be located on a nucleobase of themodified nucleic acid molecule. The modification on the nucleobase maybe selected from the group consisting of cytosine, guanine, adenine,thymine and uracil. The modification on the nucleobase may be selectedfrom the group consisting of deaza-adenosine and deaza-guanosine, andthe linker may be attached at a C-7 or C-8 position of saiddeaza-adenosine or deaza-guanosine. The modified nucleobase may beselected from the group consisting of cytosine and uracil, and thelinker may be attached to the modified nucleobase at an N-3 or C-5position. The linker attached to the nucleobase may be selected from thegroup consisting of diethylene glycol, dipropylene glycol, triethyleneglycol, tripropylene glycol, tetraethylene glycol, tetraethylene glycol,divalent alkyl, alkenyl, alkynyl moiety, ester, amide, and ether moiety.

In one embodiment, two modifications of the nucleic acid molecule may belocated on nucleosides of the modified nucleic acid molecule. Themodified nucleosides may be selected from 5-methylcytosine andpseudouridine.

In one embodiment, two modifications of the modified nucleic acidmolecule may be located on a nucleotide or a nucleoside. In oneembodiment, the present disclosure provides a formulation comprising anucleic acid molecule such as, but not limited to, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 9 and SEQ ID NO: 10 and a delivery agent. The nucleicacid molecule may comprise a polyA tail about 160 nucleotides in length.Further, the nucleic acid molecule may comprise at least one 5′ terminalcap such as, but not limited to, Cap0, Cap1, ARCA, inosine,N1-methyl-guanosine, 2′ fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine.

In one embodiment, the present disclosure provides a nucleic acid of SEQID NO: 6, a 5′ terminal cap which is Cap1, a poly A tail ofapproximately 160 nucleotides in length and a delivery agent.

In one embodiment, the present disclosure provides a nucleic acid of SEQID NO: 7, a 5′ terminal cap which is Cap1, a poly A tail ofapproximately 160 nucleotides in length and a delivery agent.

In one embodiment, the present disclosure provides a nucleic acid of SEQID NO: 9, a 5′ terminal cap which is Cap1, a poly A tail ofapproximately 160 nucleotides in length and a delivery agent.

In one embodiment, the present disclosure provides a nucleic acid of SEQID NO: 10, a 5′ terminal cap which is Cap1, a poly A tail ofapproximately 160 nucleotides in length and a delivery agent.

In one embodiment, the delivery agent comprises at least one method toimprove delivery selected from the group consisting of lipidoids,liposomes, lipid nanoparticles, rapidly eliminated lipid nanoparticles(reLNPs), polymers, lipoplexes, peptides, proteins, hydrogels, sealants,chemical modifications, conjugation, cells and enhancers. The lipidoid,lipid nanoparticle and rapidly eliminated lipid nanoparticles which maybe used as a delivery agent may include a lipid which may be selectedfrom the group consisting of C12-200, MD1, 98N12-5, DLin-DMA,DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, PLGA, PEG, PEG-DMG, PEGylatedlipids and analogs thereof. The rapidly eliminated lipid nanoparticlemay have an ester linkage at the terminal end of the lipid chain, or anester linkage may be an internal linkage located to the right or left ofa saturated carbon in the lipid chain. The rapidly eliminated lipidnanoparticle which may be used as a delivery agent may be, but is notlimited to, DLin-MC3-DMA and DLin-DMA.

In one embodiment, the lipid nanoparticle may comprise PEG and at leastone component such as, but not limited to, cholesterol, cationic lipidand fusogenic lipid.

In one embodiment, the lipid nanoparticle may comprise at least one of aPEG, cholesterol, cationic lipid and fusogenic lipid.

In one embodiment, the fusogenic lipid is disteroylphophatidyl choline(DSPC). In another embodiment, the PEG lipid is PEG-DMG. In yet anotherembodiment, the cationic lipid may be, but not limited to, DLin-DMA,DLin-MC3-DMA, C12-200, 98N12-5 and DLin-KC2-DMA.

In one embodiment, the lipid nanoparticle composition may comprise 50mol % cationic lipid, 10 mol % DSPC, 1.5-3.0 mol % PEG and 37-38.5 mol %cholesterol.

In one embodiment, a modified nucleic acid may be formulated with PLGAto form a sustained release formulation. In another embodiment, amodified nucleic acid may be formulated with PLGA and other activeand/or inactive components to form a sustained release formulation. Inone embodiment, the modified nucleic acid molecule may include, but isnot limited to, SEQ ID NO: 9 and SEQ ID NO: 10.

In one embodiment, a sustained release formulation may comprise asustained release microsphere. The sustained release microsphere may beabout 10 to about 50 um in diameter. In another embodiment, thesustained release microsphere may contain about 0.001 to about 1.0weight percent of at least one modified nucleic acid molecule.

In one embodiment, the modified nucleic acids of the present inventionmay include at least one stop codon before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the modified nucleic acids of the present invention includethe stop codon TGA and one additional stop codon. In a furtherembodiment the addition stop codon may be TAA. In another embodiment,the modified nucleic acid of the present invention includes three stopcodons.

In one embodiment, the present disclosure provides a controlled releaseformulation comprising a modified nucleic acid which may encode apolypeptide of interest. The modified nucleic acid may be encapsulatedor substantially encapsulated in a delivery agent. The delivery agentmay be coated, covered, surrounded, enclosed or comprise a layer ofpolymer, hydrogel and/or surgical sealant. In a further embodiment, thecontrolled release formulation may comprise a second layer of polymer,hydrogel and/or surgical sealant.

In one embodiment, the delivery agent of the controlled releaseformulation may include, but is not limited to, lipidoids, liposomes,lipid nanoparticles, rapidly eliminated lipid nanoparticles, lipoplexesand self-assembled lipid nanoparticles.

The polymer which may be used in the controlled release formulation mayinclude, but is not limited to, PLGA, ethylene vinyl acetate, poloxamerand GELSITE®. The surgical sealant which may be used in the controlledrelease formulation may include, but is not limited to, fibrinogenpolymers, TISSEELL®, PEG-based sealants and COSEAL®.

In one embodiment, the delivery agent of the controlled releaseformulation comprises a lipid nanoparticle or a rapidly eliminated lipidnanoparticle delivery agent. In one aspect, the lipid nanoparticle orrapidly eliminated lipid nanoparticle may be coated, substantiallycoated, covered, substantially covered, surrounded, substantiallysurrounded, enclosed, substantially enclosed or comprises a layer ofpolymer, hydrogel and/or surgical sealant. In another aspect, thedelivery agent may be a lipid nanoparticle which may be coated,substantially coated, covered, substantially covered, surrounded,substantially surrounded, enclosed, substantially enclosed or comprisesa layer of PLGA.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 illustrates lipid structures in the prior art useful in thepresent invention. Shown are the structures for 98N12-5 (TETA5-LAP),DLin-DMA, DLin-K-DMA(2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA,DLin-MC3-DMA and C12-200.

FIG. 2 is a representative plasmid useful in the IVT reactions taughtherein. The plasmid contains Insert 64818, designed by the instantinventors.

FIG. 3 is a gel profile of modified mRNA encapsulated in PLGAmicrospheres.

DETAILED DESCRIPTION

The delivery of nucleic acids into cells has many undesiredcomplications including the integration of the nucleic acid into thetarget cell genome which may result in imprecise expression levels, thedeleterious transfer of the nucleic acid to progeny and neighbor cellsand a substantial risk of causing mutations. The modified nucleic acidmolecules of the present disclosure are capable of reducing the innateimmune activity of a population of cells into which they are introduced,thus increasing the efficiency of protein production in that cellpopulation. Further, one or more additional advantageous activitiesand/or properties of the nucleic acids and proteins of the presentdisclosure are described herein.

In addition, provided herein are methods of treating a subject having orbeing suspected of having a disease, disorder and/or condition themethods comprising administering to a subject in need of such treatmenta composition described herein in an amount sufficient to treat thedisease, disorder and/or condition.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of methods featured in the invention, suitablemethods and materials are described below.

Modified Nucleic Acid Molecules

The present disclosure provides nucleic acids, including RNA such asmRNA, which contain one or more modified nucleosides or nucleotides(termed “modified nucleic acid molecules,” “modified mRNA” or “modifiedmRNA molecules”) as described herein. The modification of the nucleicacid molecules of the present invention may have useful propertiesincluding, but not limited to, a significant decrease in or a lack of asubstantial induction of the innate immune response of a cell into whichthe modified mRNA is introduced. The modified nucleic acid molecules mayalso exhibit enhanced efficiency of protein production, intracellularretention of nucleic acids, and viability of contacted cells, as well ashaving reduced immunogenicity as compared to unmodified nucleic acidmolecules.

Provided are modified nucleic acid molecules containing a translatableregion and one, two, or more than two different nucleoside modificationsExemplary nucleic acids for use in this disclosure include ribonucleicacids (RNA), deoxyribonucleic acids (DNAs), threose nucleic acids(TNAs), glycol nucleic acids (GNAs), locked nucleic acids (LNAs) or ahybrid thereof. In preferred embodiments, the modified nucleic acidmolecules include messenger RNA (mRNA). As described herein, themodified nucleic acid molecules of the present disclosure may notsubstantially induce an innate immune response of a cell into which themodified mRNA is introduced. In another embodiment, the modified nucleicacid molecule may exhibit reduced degradation, as compared to a nucleicacid that has not been modified, in a cell where the modified nucleicacid molecule is introduced.

The term “nucleic acid” includes any compound and/or substance that isor can be incorporated into an oligonucleotide chain. Exemplary nucleicacids for use in accordance with the present disclosure include, but arenot limited to, one or more of DNA, cDNA, RNA including messenger RNA(mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA,shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that inducetriple helix formation, aptamers, vectors and the like.

In certain embodiments, it is desirable to intracellularly degrade amodified nucleic acid molecule introduced into the cell. For example itwould be desirable to degrade a modified nucleic acid molecule ifprecise timing of protein production was desired. Thus, the presentdisclosure provides a modified nucleic acid molecule containing adegradation domain, which is capable of being acted on in a directedmanner within a cell.

In some embodiments, the modified nucleic acid molecules may bechemically modified on the sugar, nucleobase (e.g., in the 5′ positionof the nucleobase), or phosphate backbone (e.g., replacing the phosphatewith another moiety such as a thiophospate). In some embodiments, themodification may result in a disruption of a major groove bindingpartner interaction, which may contribute to an innate immune response.In some embodiments, the formulation composition, when administered to asubject, can result in improved bioavailability, therapeutic window, orvolume of distribution of the modified nucleic acid molecule relative toadministration of the modified nucleic acid molecule without theincorporation of the delivery agent. In some embodiments, the modifiednucleosides and nucleotides of the modified nucleic acid molecules ofthe present invention may be synthesized using the O-protected compoundsdescribed in International Pub. No. WO2012138530, the contents of whichis herein incorporated by reference in its entirety.

In certain embodiments, the modified nucleic acid molecule may comprisemRNA. In particular embodiments, the modified mRNA (mmRNA) may bederived from cDNA. In certain embodiments, mmRNA may comprise at leasttwo nucleoside modifications. In one embodiment, the nucleosidemodifications may be selected from 5-methylcytosine and pseudouridine.In another embodiment, at least one of the nucleoside modifications isnot 5-methylcytosine and/or pseudouridine. In certain embodiments thedelivery agent may comprise formulations allowing for localized andsystemic delivery of mmRNA. The formulations of the modified nucleicacids molecules and/or mmRNA may be selected from, but are not limitedto, lipidoids, liposomes and lipid nanoparticles, rapidly eliminatedlipid nanoparticles, polymers, lipoplexes, peptides and proteins, atleast one chemical modification and conjugation, enhancers, and/orcells.

In one embodiment, the modified nucleic acid molecules of the presentinvention may include at least two stop codons before the 3′untranslated region (UTR). The stop codon may be selected from TGA, TAAand TAG. In one embodiment, the nucleic acids of the present inventioninclude the stop codon TGA and one additional stop codon. In a furtherembodiment the addition stop codon may be TAA. In another embodiment,the modified nucleic acid molecules may comprise three stop codons.

Other components of a nucleic acid are optional in a modified nucleicacid molecule but these components may be beneficial in someembodiments.

Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′ UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′ UTR starts immediately following a stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into the modified mRNAmolecules of the present invention to enhance the stability of themolecule. The specific features can also be incorporated to ensurecontrolled down-regulation of the transcript in case they aremisdirected to undesired organs sites.

5′ UTR and Translation Initiation

Natural 5′ UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG (SEQ ID NO: 1), where R is a purine (adenine or guanine)three bases upstream of the start codon (AUG), which is followed byanother ‘G’. 5′ UTR also have been known to form secondary structureswhich are involved in elongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the modified mRNA molecules of the invention. Forexample, introduction of 5′ UTR of liver-expressed mRNA, such asalbumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alphafetoprotein, erythropoietin, or Factor VIII, could be used to enhanceexpression of a modified nucleic acid molecule, such as a mmRNA, inhepatic cell lines or liver. Likewise, use of 5′ UTR from othertissue-specific mRNA to improve expression in that tissue is possiblefor muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), forendothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF,GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), foradipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lungepithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR) UTRsof the modified nucleic acid molecules of the present invention. Forexample, introns or portions of introns sequences may be incorporatedinto the flanking regions of the modified mRNA of the invention.Incorporation of intronic sequences may increase protein production aswell as mRNA levels.

3′ UTR and the AU Rich Elements

3′ UTRs are known to have stretches of Adenosines and Uridines embeddedin them. These AU rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU rich elements (AREs) can be separated intothree classes (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) (SEQ ID NO: 2) nonamers. Molecules containing thistype of AREs include GM-CSF and TNF-a. Class III ARES are less welldefined. These U rich regions do not contain an AUUUA motif. c-Jun andMyogenin are two well-studied examples of this class. Most proteinsbinding to the AREs are known to destabilize the messenger, whereasmembers of the ELAV family, most notably HuR, have been documented toincrease the stability of mRNA. HuR binds to AREs of all the threeclasses. Engineering the HuR specific binding sites into the 3′ UTR ofnucleic acid molecules will lead to HuR binding and thus, stabilizationof the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of modified mRNA of the invention.When engineering specific modified mRNA, one or more copies of an AREcan be introduced to make modified mRNA of the invention less stable andthereby curtail translation and decrease production of the resultantprotein.

Likewise, AREs can be identified and removed or mutated to increase theintracellular stability and thus increase translation and production ofthe resultant protein. Transfection experiments can be conducted inrelevant cell lines, using modified mRNA of the invention and proteinproduction can be assayed at various time points post-transfection. Forexample, cells can be transfected with different ARE-engineeringmolecules and by using an ELISA kit to the relevant protein and assayingprotein produced at 6 hours, 12 hours, 24 hours, 48 hours, and 7 dayspost-transfection.

Incorporating microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′ UTR of nucleic acid molecules and down-regulate geneexpression either by reducing nucleic acid molecule stability or byinhibiting translation. The modified mRNA of the invention may compriseone or more microRNA target sequences, microRNA sequences, or microRNAseeds. Such sequences may correspond to any known microRNA such as thosetaught in US Publication US2005/0261218 and US PublicationUS2005/0059005, the contents of which are incorporated herein byreference in their entirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked byan adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol. Cell. 2007 Jul. 6; 27(1):91-105; each of which isherein incorporated by reference in their entirety. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of modified mRNA ofthe invention one can target the molecule for degradation or reducedtranslation, provided the microRNA in question is available. Thisprocess will reduce the hazard of off target effects upon nucleic acidmolecule delivery. Identification of microRNA, microRNA target regions,and their expression patterns and role in biology have been reported(Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and ChereshCurr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 201226:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; each of which isherein incorporated by reference in its entirety).

For example, if the modified nucleic acid molecule is a modified mRNAand is not intended to be delivered to the liver but ends up there, thenmiR-122, a microRNA abundant in liver, can inhibit the expression of thegene of interest if one or multiple target sites of miR-122 areengineered into the 3′ UTR of the modified mRNA. Introduction of one ormultiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of amodified nucleic acid molecule and/or modified mRNA.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the modified mRNA of the presentinvention, microRNA binding sites can be engineered out of (i.e. removedfrom) sequences in which they naturally occur in order to increaseprotein expression in specific tissues. For example, miR-122 bindingsites may be removed to improve protein expression in the liver.Regulation of expression in multiple tissues can be accomplished throughintroduction or removal or one or several microRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176; herein incorporated byreference in its entirety). In the modified mRNA of the presentinvention, binding sites for microRNAs that are involved in suchprocesses may be removed or introduced, in order to tailor theexpression of the modified mRNA expression to biologically relevant celltypes or to the context of relevant biological processes.

Lastly, through an understanding of the expression patterns of microRNAin different cell types, modified mRNA can be engineered for moretargeted expression in specific cell types or only under specificbiological conditions. Through introduction of tissue-specific microRNAbinding sites, modified mRNA could be designed that would be optimal forprotein expression in a tissue or in the context of a biologicalcondition.

Transfection experiments can be conducted in relevant cell lines, usingengineered modified mRNA and protein production can be assayed atvarious time points post-transfection. For example, cells can betransfected with different microRNA binding site-engineering modifiedmRNA and by using an ELISA kit to the relevant protein and assayingprotein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 hour and 7days post-transfection. In vivo experiments can also be conducted usingmicroRNA-binding site-engineered molecules to examine changes intissue-specific expression of formulated modified mRNA.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the modified mRNA of the present invention may generatea non-hydrolyzable cap structure preventing decapping and thusincreasing mRNA half-life. Because cap structure hydrolysis requirescleavage of 5′-ppp-5′ phosphorodiester linkages, modified nucleotidesmay be used during the capping reaction. For example, a Vaccinia CappingEnzyme from New England Biolabs (Ipswich, Mass.) may be used withα-thio-guanosine nucleotides according to the manufacturer'sinstructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.Additional modified guanosine nucleotides may be used such asα-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/or linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′ mppp-G;which may equivalently be designated 3′ O-Me-m7G(5)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methlyated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptscan remain uncapped. This, as well as the structural differences of acap analog from an endogenous 5′-cap structures of nucleic acidsproduced by the endogenous, cellular transcription machinery, may leadto reduced translational competency and reduced cellular stability.

Modified mRNA of the present invention may also be cappedpost-transcriptionally, using enzymes, in order to generate moreauthentic 5′-cap structures. As used herein, the phrase “more authentic”refers to a feature that closely mirrors or mimics, either structurallyor functionally, an endogenous or wild type feature. That is, a “moreauthentic” feature is better representative of an endogenous, wild-type,natural or physiological cellular function and/or structure as comparedto synthetic features or analogs, etc., of the prior art, or whichoutperforms the corresponding endogenous, wild-type, natural orphysiological feature in one or more respects. Non-limiting examples ofmore authentic 5′ cap structures of the present invention are thosewhich, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural orphysiological 5′ cap structure). For example, recombinant Vaccinia VirusCapping Enzyme and recombinant 2′-O-methyltransferase enzyme can createa canonical 5′-5′-triphosphate linkage between the 5′-terminalnucleotide of an mRNA and a guanine cap nucleotide wherein the capguanine contains an N7 methylation and the 5′-terminal nucleotide of themRNA contains a 2′-O-methyl. Such a structure is termed the Cap1structure. This cap results in a higher translational-competency andcellular stability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′ cap analog structures known inthe art. Cap structures include, but are not limited to,7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)N1mpNp (cap 1), and7mG(5′)-ppp(5′)N1mpN2mp (cap 2).

Because the modified mRNA may be capped post-transcriptionally, andbecause this process is more efficient, nearly 100% of the modified mRNAmay be capped. This is in contrast to ˜80% when a cap analog is linkedto an mRNA in the course of an in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,LNA-guanosine, and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can beengineered and inserted in the 3′ UTR of the modified mRNA of theinvention and can stimulate the translation of the mRNA in vitro and invivo. Transfection experiments can be conducted in relevant cell linesat and protein production can be assayed by ELISA at 12 hour, 24 hour,48 hour, 72 hour and day 7 post-transfection.

IRES Sequences

Further, provided are modified mRNA which may contain an internalribosome entry site (IRES). First identified as a feature Picorna virusRNA, IRES plays an important role in initiating protein synthesis inabsence of the 5′ cap structure. An IRES may act as the sole ribosomebinding site, or may serve as one of multiple ribosome binding sites ofan mRNA. Modified mRNA containing more than one functional ribosomebinding site may encode several peptides or polypeptides that aretranslated independently by the ribosomes (“multicistronic nucleic acidmolecules”). When modified mRNA are provided with an IRES, furtheroptionally provided is a second translatable region. Examples of IRESsequences that can be used according to the invention include withoutlimitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV),polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouthdisease viruses (FMDV), hepatitis C viruses (HCV), classical swine feverviruses (CSFV), murine leukemia virus (MLV), simian immune deficiencyviruses (SIV) or cricket paralysis viruses (CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a modified nucleic acid molecule such as a modified mRNAmolecules in order to increase stability. Immediately aftertranscription, the 3′ end of the transcript may be cleaved to free a 3′hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides tothe RNA. The process, called polyadenylation, adds a poly-A tail thatcan be between, for example, approximately 100 and 250 residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the modified mRNA of the present invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000 nucleotides). In some embodiments, the modified mRNA includesfrom about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000,from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000,from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500,from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000,from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall modified mRNA. This design may be based on the length of thecoding region, the length of a particular feature or region (such as theflanking regions), or based on the length of the ultimate productexpressed from the modified mRNA.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the modified mRNA, region or featurethereof. The poly-A tail may also be designed as a fraction of modifiedmRNA to which it belongs. In this context, the poly-A tail may be 10,20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of themolecule or the total length of the molecule minus the poly-A tail.Further, engineered binding sites and conjugation of modified mRNA forPoly-A binding protein may enhance expression.

Additionally, multiple distinct modified mRNA may be linked together tothe PABP (Poly-A binding protein) through the 3′-end using modifiednucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hour, 24 hour, 48 hour, 72 hourand day 7 post-transfection.

In one embodiment, the modified mRNA of the present invention aredesigned to include a polyA-G Quartet. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultant mmRNAmolecule is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein productionequivalent to at least 75% of that seen using a poly-A tail of 120nucleotides alone.

Modifications

The modified nucleic acids and modified mRNA (mmRNA) of the inventionmay contain one, two, or more different modifications. In someembodiments, modified nucleic acids and mmRNA may contain one, two, ormore different nucleoside or nucleotide modifications. In someembodiments, a modified nucleic acid or mmRNA (e.g., having one or moremmRNA molecules) introduced to a cell may exhibit reduced degradation inthe cell, as compared to an unmodified nucleic acid or mmRNA.

The modified nucleic acids and mmRNA can include any usefulmodification, such as to the sugar, the nucleobase (e.g., one or moremodifications of a nucleobase, such as by replacing or substituting anatom of a pyrimidine nucleobase with optionally substituted amino,optionally substituted thiol, optionally substituted alkyl (e.g., methylor ethyl), or halo (e.g., chloro or fluoro), or the internucleosidelinkage (e.g., one or more modification to the phosphodiester backbone).In certain embodiments, modifications are present in both the sugar andthe internucleoside linkage (e.g., one or modifications, such as thosepresent in ribonucleic acids (RNA), deoxyribonucleic acids (DNAs),threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptidenucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof).Additional modifications are described herein.

As described herein, the modified nucleic acids and mmRNA of theinvention do not substantially induce an innate immune response of acell into which the mRNA is introduced. In certain embodiments, it maydesirable to intracellularly degrade a modified nucleic acid molecule ormodified nucleic acid molecule introduced into the cell. For example,degradation of a modified nucleic acid molecule or modified mRNA may bepreferable if precise timing of protein production is desired. Thus, insome embodiments, the invention provides a modified nucleic acidmolecule containing a degradation domain, which is capable of beingacted on in a directed manner within a cell. In another aspect, thepresent disclosure provides nucleic acids comprising a nucleoside ornucleotide that can disrupt the binding of a major groove interacting,e.g. binding, partner with the nucleic acid (e.g., where the modifiednucleotide has decreased binding affinity to major groove interactingpartner, as compared to an unmodified nucleotide).

The modified nucleic acid and mmRNA can optionally include other agents(e.g., RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisenseRNA, ribozymes, catalytic DNA, tRNA, RNA that induce triple helixformation, aptamers, vectors, etc.). In some embodiments, the modifiednucleic acids or mmRNA may include one or more messenger RNA (mRNA) andone or more modified nucleoside or nucleotides (e.g., mmRNA molecules).Details for these modified nucleic acids and mmRNA follow.

Modified Nucleic Acids

The modified nucleic acids or mmRNA of the invention may include a firstregion of linked nucleosides encoding a polypeptide of interest, a firstflanking region located at the 5′ terminus of the first region, and asecond flanking region located at the 3′ terminus of the first region.

In some embodiments, the modified nucleic acids or mmRNA includes nnumber of linked nucleosides having Formula (Ia) or Formula (Ia-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R¹, R², R³, R⁴, and R⁵ is ifpresent, independently, H, halo, hydroxy, thiol, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R^(1′),R^(1″), R^(2′)R^(2″), or R⁵ (e.g., the combination of R^(1′) and R³, thecombination of R^(1″) and R³, the combination of R^(2′) and R³, thecombination of R^(2″) and R³, or the combination of R⁵ and R³) can jointogether to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein thecombination of R⁵ with one or more of R^(1′), R^(1″), R^(2′), or R^(2″)(e.g., the combination of R^(1′) and R⁵, the combination of R^(1″) andR⁵, the combination of R^(2′) and R⁵, or the combination of R^(2″) andR⁵) can join together to form optionally substituted alkylene oroptionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl);and wherein the combination of R⁴ and one or more of R^(1′), R^(1″),R^(2′), R^(2″), R³, or R⁵ can join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl);

each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof), wherein the combination of B and R^(1′), the combination of Band R^(2′), the combination of B and R^(1″), or the combination of B andR^(2″) can, taken together with the carbons to which they are attached,optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) orwherein the combination of B, R^(1″), and R³ or the combination of B,R^(2″), and R³ can optionally form a tricyclic or tetracyclic group(e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula(IIo)-(IIp) herein). In some embodiments, the modified nucleic acid ormmRNA includes a modified ribose.

In some embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (Ia-2)-(Ia-5) or apharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (Ib) or Formula (Ib-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of R¹, R^(3′), R^(3″), and R⁴ is, independently, H, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionallysubstituted hydroxyalkoxy, optionally substituted amino, azido,optionally substituted aryl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, or absent; and wherein the combination of R¹ and R^(3′) orthe combination of R¹ and R^(3″) can be taken together to formoptionally substituted alkylene or optionally substituted heteroalkylene(e.g., to produce a locked nucleic acid);

each R⁵ is, independently, H, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, or absent;

each of Y¹, Y², and Y³ is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl; each Y⁴ is, independently, H, hydroxy,thiol, boranyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted alkoxy,optionally substituted alkenyloxy, optionally substituted alkynyloxy,optionally substituted alkoxyalkoxy, or optionally substituted amino;

n is an integer from 1 to 100,000; and

B is a nucleobase.

In some embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (Ic):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of B¹, B², and B³ is, independently, a nucleobase (e.g., a purine,a pyrimidine, or derivatives thereof, as described herein), H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl, wherein one and only one of B¹, B²,and B³ is a nucleobase;

each of R^(b1), R^(b2), R^(b3), R³, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl oroptionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

wherein the ring including U can include one or more double bonds.

In particular embodiments, the ring including U does not have a doublebond between U—CB³R^(b3) or between CB³R^(b3)—C^(B2)R^(b2).

In some embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (Id):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

each R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, optionally substituted alkylene (e.g.,methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the modified nucleic acid molecules or modifiedmRNA includes n number of linked nucleosides having Formula (Ie):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of U′ and U″ is, independently, O, S, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl;

each R⁶ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each Y^(5′) is, independently, O, S, optionally substituted alkylene(e.g., methylene or ethylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (If) or (If-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of U′ and U″ is, independently, O, S, N,N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl (e.g., U′ is Oand U″ is N);

- - - is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R³, and R⁴ is, independently, H,halo, hydroxy, thiol, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,optionally substituted amino, azido, optionally substituted aryl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, or absent; and wherein thecombination of R^(1′) and R³, the combination of R^(1″) and R³, thecombination of R^(2′) and R³, or the combination of R^(2″) and R³ can betaken together to form optionally substituted alkylene or optionallysubstituted heteroalkylene (e.g., to produce a locked nucleic acid);each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments of the modified nucleic acid or mmRNA (e.g.,(Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), thering including U has one or two double bonds.

In some embodiments of the modified nucleic acid or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), eachof R¹, R^(1″), and R^(1″), if present, is H. In further embodiments,each of R², R^(2′), and R^(2″), if present, is, independently, H, halo(e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In particularembodiments, alkoxyalkoxy is —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl). In some embodiments, s2 is 0, s1 is 1 or 2,s3 is 0 or 1, and R′ is C₁₋₆ alkyl.

In some embodiments of the modified nucleic acid or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), eachof R², R^(2′), and R^(2″), if present, is H. In further embodiments,each of R¹, R^(1′), and R^(1″), if present, is, independently, H, halo(e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In particularembodiments, alkoxyalkoxy is —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl). In some embodiments, s2 is 0, s1 is 1 or 2,s3 is 0 or 1, and R′ is C₁₋₆ alkyl.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), eachof R³, R⁴, and R⁵ is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkyl, optionally substituted alkoxy (e.g.,methoxy or ethoxy), or optionally substituted alkoxyalkoxy. Inparticular embodiments, R³ is H, R⁴ is H, R⁵ is H, or R³, R⁴, and R⁵ areall H. In particular embodiments, R³ is C₁₋₆ alkyl, R⁴ is C₁₋₆ alkyl, R⁵is C₁₋₆ alkyl, or R³, R⁴, and R⁵ are all C₁₋₆ alkyl. In particularembodiments, R³ and R⁴ are both H, and R⁵ is C₁₋₆ alkyl.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R³ andR⁵ join together to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl, such as trans-3′,4′analogs, wherein R³ and R⁵ join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R³ andone or more of R^(1′), R^(1″), R^(2′), R^(2″), or R⁵ join together toform optionally substituted alkylene or optionally substitutedheteroalkylene and, taken together with the carbons to which they areattached, provide an optionally substituted heterocyclyl (e.g., abicyclic, tricyclic, or tetracyclic heterocyclyl, R³ and one or more ofR^(1′), R^(1″), R^(2′), R^(2″), or R⁵ join together to formheteroalkylene (e.g., —(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein eachof b1, b2, and b3 are, independently, an integer from 0 to 3).

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R⁵ andone or more of R^(1′), R^(1″), R^(2′), or R^(2″) join together to formoptionally substituted alkylene or optionally substituted heteroalkyleneand, taken together with the carbons to which they are attached, providean optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl, R⁵ and one or more of R^(1′), R^(1″), R^(2′),or R^(2″) join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), eachY² is, independently, O, S, or —NR^(N1)—, wherein R^(N1) is H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl. In particularembodiments, Y² is NR^(N1)—, wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl,or n-propyl).

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), eachY³ is, independently, O or S.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R¹ isH; each R² is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is0 or 1, and R′ is C₁₋₆ alkyl); each Y² is, independently, O or—NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl (e.g., wherein R^(N1) is H or optionally substitutedalkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl, orn-propyl)); and each Y³ is, independently, O or S (e.g., S). In furtherembodiments, R³ is H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In yet furtherembodiments, each Y¹ is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y⁴ is,independently, H, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted thioalkoxy,optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), eachR¹ is, independently, H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each ofs2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H orC₁₋₂₀ alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl); R² is H; each Y² is, independently, O or —NR^(N1)—,wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl (e.g., wherein R^(N1) is H or optionally substitutedalkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl, orn-propyl)); and each Y³ is, independently, O or S (e.g., S). In furtherembodiments, R³ is H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In yet furtherembodiments, each Y¹ is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y⁴ is,independently, H, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted thioalkoxy,optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), thering including U is in the β-D (e.g., β-D-ribo) configuration.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), thering including U is in the α-L (e.g., α-L-ribo) configuration.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), one ormore B is not pseudouridine (ψ) or 5-methyl-cytidine (m⁵C). In someembodiments, about 10% to about 100% of B nucleobases is not ψ or m⁵C(e.g., from 10% to 20%, from 10% to 35%, from 10% to 50%, from 10% to60%, from 10% to 75%, from 10% to 90%, from 10% to 95%, from 10% to 98%,from 10% to 99%, from 20% to 35%, from 20% to 50%, from 20% to 60%, from20% to 75%, from 20% to 90%, from 20% to 95%, from 20% to 98%, from 20%to 99%, from 20% to 100%, from 50% to 60%, from 50% to 75%, from 50% to90%, from 50% to 95%, from 50% to 98%, from 50% to 99%, from 50% to100%, from 75% to 90%, from 75% to 95%, from 75% to 98%, from 75% to99%, and from 75% to 100% of n number of B is not ψ or m⁵C). In someembodiments, B is not ψ or m⁵C.

In some embodiments of the modified nucleic acids or mmRNA (e.g.,Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), when Bis an unmodified nucleobase selected from cytosine, guanine, uracil andadenine, then at least one of Y¹, Y², or Y³ is not O.

In some embodiments, the modified nucleic acids or mmRNA includes amodified ribose. In some embodiments, modified nucleic acids or mmRNAincludes n number of linked nucleosides having Formula (IIa)-(IIc):

or a pharmaceutically acceptable salt or stereoisomer thereof. Inparticular embodiments, U is O or C(R^(U))_(nu), wherein nu is aninteger from 0 to 2 and each R^(U) is, independently, H, halo, oroptionally substituted alkyl (e.g., U is —CH₂— or —CH—). In otherembodiments, each of R¹, R², R³, R⁴, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or absent (e.g., each R¹ and R² is,independently, H, halo, hydroxy, optionally substituted alkyl, oroptionally substituted alkoxy; each R³ and R⁴ is, independently, H oroptionally substituted alkyl; and R⁵ is H or hydroxy), and - - - is asingle bond or double bond.

In particular embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (IIb-1)-(IIb-2):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In other embodiments, each of R¹ andR² is, independently, H, halo, hydroxy, thiol, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy). In particular embodiments, R² is hydroxy oroptionally substituted alkoxy (e.g., methoxy, ethoxy, or any describedherein).

In particular embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (IIc-1)-(IIc-4):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In some embodiments, each of R¹, R²,and R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy; and each R³ is, independently, H oroptionally substituted alkyl)). In particular embodiments, R² isoptionally substituted alkoxy (e.g., methoxy or ethoxy, or any describedherein). In particular embodiments, R¹ is optionally substituted alkyl,and R² is hydroxy. In other embodiments, R¹ is hydroxy, and R² isoptionally substituted alkyl. In further embodiments, R³ is optionallysubstituted alkyl.

In some embodiments, the modified nucleic acids or mmRNA includes anacyclic modified ribose. In some embodiments, the modified nucleic acidsor mmRNA includes n number of linked nucleosides having Formula(IId)-(IIf):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the modified nucleic acids or mmRNA includes anacyclic modified hexitol. In some embodiments, the modified nucleicacids or mmRNA includes n number of linked nucleosides having Formula(IIg)-(IIj):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the modified nucleic acids or mmRNA includes asugar moiety having a contracted or an expanded ribose ring. In someembodiments, the modified nucleic acids or mmRNA includes n number oflinked nucleosides having Formula (IIk)-(IIm):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of R^(1′), R^(1″), R^(2′), and R^(2″) is, independently, H, halo,hydroxy, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted alkenyloxy, optionally substituted alkynyloxy,optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy,or absent; and wherein the combination of R^(2′) and R³ or thecombination of R^(2″) and R³ can be taken together to form optionallysubstituted alkylene or optionally substituted heteroalkylene.

In some embodiments, the modified nucleic acids or mmRNA includes alocked modified ribose. In some embodiments, the modified nucleic acidsor mmRNA includes n number of linked nucleosides having Formula (IIn):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the modified nucleic acid or mmRNA includes nnumber of linked nucleosides having Formula (IIn-1)-(II-n2):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂₋₅ or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂₋₅—CH₂CH₂₋₅ or —CH₂CH₂CH₂—)).

In some embodiments, the modified nucleic acids or mmRNA includes alocked modified ribose that forms a tetracyclic heterocyclyl. In someembodiments, the modified nucleic acids or mmRNA includes n number oflinked nucleosides having Formula (IIo):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(12a), R^(12c), T^(1′), T^(1″), T^(2′), T^(2″), V¹, and V³ are asdescribed herein.

Any of the formulas for the modified nucleic acids or mmRNA can includeone or more nucleobases described herein (e.g., Formulas (b1)-(b43)).

In one embodiment, the present invention provides methods of preparing amodified nucleic acids or mmRNA comprising at least one nucleotide(e.g., mmRNA molecule), wherein the modified nucleic acid comprises nnumber of nucleosides having Formula (Ia), as defined herein:

the method comprising reacting a compound of Formula (IIIa), as definedherein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a modified nucleic acids or mmRNA comprising at least onenucleotide (e.g., mmRNA molecule), the method comprising: reacting acompound of Formula (IIIa), as defined herein, with a primer, a cDNAtemplate, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing amodified nucleic acids or mmRNA comprising at least one nucleotide(e.g., mmRNA molecule), wherein the modified nucleic acid comprises nnumber of nucleosides having Formula (Ia-1), as defined herein:

the method comprising reacting a compound of Formula (IIIa-1), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a modified nucleic acids or mmRNA comprising at least onenucleotide (e.g., mmRNA molecule), the method comprising reacting acompound of Formula (IIIa-1), as defined herein, with a primer, a cDNAtemplate, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing amodified mRNA comprising at least one nucleotide (e.g., mmRNA molecule),wherein the polynucleotide comprises n number of nucleosides havingFormula (Ia-2), as defined herein:

the method comprising reacting a compound of Formula (IIIa-2), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a modified mRNA comprising at least one nucleotide (e.g.,mmRNA molecule), the method comprising:

reacting a compound of Formula (IIIa-2), as defined herein, with aprimer, a cDNA template, and an RNA polymerase.

In some embodiments, the reaction may be repeated from 1 to about 7,000times. In any of the embodiments herein, B may be a nucleobase ofFormula (b1)-(b43).

The modified nucleic acids and mmRNA can optionally include 5′ and/or 3′flanking regions, which are described herein.

Modified RNA (e.g. mmRNA) Molecules

The present invention also includes building blocks, e.g., modifiedribonucleosides, modified ribonucleotides, of modified RNA (mmRNA)molecules. For example, these mmRNA can be useful for preparing themodified nucleic acids or mmRNA of the invention.

In some embodiments, the building block molecule has Formula (IIIa) or(IIIa-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinthe substituents are as described herein (e.g., for Formula (Ia) and(Ia-1)), and wherein when B is an unmodified nucleobase selected fromcytosine, guanine, uracil and adenine, then at least one of Y¹, Y², orY³ is not O.

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid or mmRNA, has Formula(IVa)-(IVb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified uracil(e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), suchas formula (b1), (b8), (b28), (b29), or (b30)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified cytosine(e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36),such as formula (b10) or (b32)). In particular embodiments, Formula(IVa) or (IVb) is combined with a modified guanine (e.g., any one offormulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,Formula (IVa) or (IVb) is combined with a modified adenine (e.g., anyone of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, has Formula(IVc)-(IVk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IVc)-(IVk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IVc)-(IVk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IVc)-(IVk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IVc)-(IVk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, has Formula(Va) or (Vb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, has Formula(IXa)-(IXd):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, has Formula(IXe)-(IXg):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, has Formula(IXh)-(IXk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, has Formula(IXl)-(IXr):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r1 and r2 is, independently, an integer from 0 to 5 (e.g., from 0to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g.,any one of (b1)-(b43)). In particular embodiments, one of Formulas(IXl)-(IXr) is combined with a modified uracil (e.g., any one offormulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),(b8), (b28), (b29), or (b30)). In particular embodiments, one ofFormulas (IXl)-(IXr) is combined with a modified cytosine (e.g., any oneof formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula(b10) or (b32)). In particular embodiments, one of Formulas (IXl)-(IXr)is combined with a modified guanine (e.g., any one of formulas(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas(IXl)-(IXr) is combined with a modified adenine (e.g., any one offormulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecules or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5) and s1 is as described herein.

In some embodiments, the building block molecule, which may beincorporated into a nucleic acid (e.g., RNA, mRNA, or mmRNA), is amodified uridine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, is amodified cytidine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)). For example, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, is amodified adenosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a modified nucleic acid molecule or mmRNA, is amodified guanosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the chemical modification can include replacementof C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, suchas cytosine or uracil) with N (e.g., replacement of the >CH group at C-5with >NR^(N1) group, wherein R^(N)1 is H or optionally substitutedalkyl). For example, the mmRNA molecule, which may be incorporated intoa modified nucleic acid molecule or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In another embodiment, the chemical modification can include replacementof the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) oroptionally substituted alkyl (e.g., methyl). For example, the mmRNAmolecule, which may be incorporated into a modified nucleic acid ormmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In yet a further embodiment, the chemical modification can include afused ring that is formed by the NH₂ at the C-4 position and the carbonatom at the C-5 position. For example, the building block molecule,which may be incorporated into a modified nucleic acid molecule ormmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which may be incorporated into a modified nucleic acid ormmRNA (e.g., RNA or mRNA, as described herein), can be modified on thesugar of the ribonucleic acid. For example, the 2′ hydroxyl group (OH)can be modified or replaced with a number of different substituents.Exemplary substitutions at the 2′-position include, but are not limitedto, H, halo, optionally substituted C₁₋₆ alkyl; optionally substitutedC₁₋₆ alkoxy; optionally substituted C₆₋₁₀ aryloxy; optionallysubstituted C₃₋₈ cycloalkyl; optionally substituted C₃₋₈ cycloalkoxy;optionally substituted C₆₋₁₀ aryloxy; optionally substituted C₆₋₁₀aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy; asugar (e.g., ribose, pentose, or any described herein); apolyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H oroptionally substituted alkyl, and n is an integer from 0 to 20 (e.g.,from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10,from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in whichthe 2′-hydroxyl is connected by a C₁₋₆ alkylene or C₁₋₆ heteroalkylenebridge to the 4′-carbon of the same ribose sugar, where exemplarybridges included methylene, propylene, ether, or amino bridges;aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino asdefined herein; and amino acid, as defined herein.

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a modified nucleic acidmolecule or mmRNA can include nucleotides containing, e.g., arabinose,as the sugar.

Modifications on the Nucleobase

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof. As described herein, “nucleotide”is defined as a nucleoside including a phosphate group. The modifiednucleotides (e.g., modified mRNA) may by synthesized by any usefulmethod, as described herein (e.g., chemically, enzymatically, orrecombinantly to include one or more modified or non-naturalnucleosides).

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine. These nucleobases can be modified orwholly replaced to provide modified nucleic acids or mmRNA moleculeshaving enhanced properties, e.g., resistance to nucleases throughdisruption of the binding of a major groove binding partner. Table 1below identifies the chemical faces of each canonical nucleotide.Circles identify the atoms comprising the respective chemical regions.

TABLE 1 Watson-Crick Major Groove Minor Groove Base-pairing Face FaceFace Pyrimidines Cytidine:

Uridine:

Purines Adenosine:

Guanosine:

In some embodiments, B is a modified uracil. Exemplary modified uracilsinclude those having Formula (b1)-(b5):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) or the combination of T^(2′) and T^(2″) join together (e.g., asin T²) to form O (oxo), S (thio), or Se (seleno);

each of V¹ and V² is, independently, O, S, N(R^(Vb))_(nv), orC(R^(Vb))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vb) is,independently, H, halo, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g.,trifluoroacetyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, or optionally substituted alkoxycarbonylalkoxy(e.g., optionally substituted with any substituent described herein,such as those selected from (1)-(21) for alkyl);

R¹⁰ is H, halo, optionally substituted amino acid, hydroxy, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aminoalkyl, optionallysubstituted hydroxyalkyl, optionally substituted hydroxyalkenyl,optionally substituted hydroxyalkynyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl;

R¹¹ is H or optionally substituted alkyl;

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, or optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy), optionally substituted carboxyalkoxy,optionally substituted carboxyaminoalkyl, or optionally substitutedcarbamoylalkyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl.

Other exemplary modified uracils include those having Formula (b6)-(b9):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) join together (e.g., as in T¹) or the combination of T^(2′) andT^(2″) join together (e.g., as in T²) to form O (oxo), S (thio), or Se(seleno), or each T¹ and T² is, independently, O (oxo), S (thio), or Se(seleno);

each of W¹ and W² is, independently, N(R^(Wa))_(nv) or C(R^(Wa))_(nv),wherein nv is an integer from 0 to 2 and each R^(Wa) is, independently,H, optionally substituted alkyl, or optionally substituted alkoxy;

each V³ is, independently, O, S, N(R^(Va))_(nv), or C(R^(Va))_(nv),wherein nv is an integer from 0 to 2 and each R^(Va) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), and wherein R^(Va) and R^(12c)taken together with the carbon atoms to which they are attached can formoptionally substituted cycloalkyl, optionally substituted aryl, oroptionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy and/or an O-protecting group), optionallysubstituted carboxyalkoxy, optionally substituted carboxyaminoalkyl,optionally substituted carbamoylalkyl, or absent;

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted alkaryl, optionally substitutedheterocyclyl, optionally substituted alkheterocyclyl, optionallysubstituted amino acid, optionally substituted alkoxycarbonylacyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedalkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,optionally substituted alkoxycarbonylalkynyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl,

wherein the combination of R^(12b) and T^(1′) or the combination ofR^(12b) and R^(12c) can join together to form optionally substitutedheterocyclyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl.

Further exemplary modified uracils include those having Formula(b28)-(b31):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T¹ and T² is, independently, O (oxo), S (thio), or Se (seleno);

each R^(Vb′) and R^(Vb″) is, independently, H, halo, optionallysubstituted amino acid, optionally substituted alkyl, optionallysubstituted haloalkyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl) (e.g., R^(Vb″) is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted aminoalkyl, e.g., substituted with an N-protecting group,such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);

R^(12a) is H, optionally substituted alkyl, optionally substitutedcarboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, or optionally substituted aminoalkynyl; and

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substituted aminoalkynyl(e.g., e.g., substituted with an N-protecting group, such as anydescribed herein, e.g., trifluoroacetyl, or sulfoalkyl),

optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl.

In particular embodiments, T¹ is O (oxo), and T² is S (thio) or Se(seleno). In other embodiments, T¹ is S (thio), and T² is O (oxo) or Se(seleno). In some embodiments, R^(Vb′) is H, optionally substitutedalkyl, or optionally substituted alkoxy.

In other embodiments, each R^(12a) and R^(12b) is, independently, H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted hydroxyalkyl. Inparticular embodiments, R^(12a) is H. In other embodiments, both R^(12a)and R^(12b) are H.

In some embodiments, each R^(Vb′) of R^(12b) is, independently,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g., trifluoroacetyl,or sulfoalkyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or optionally substituted acylaminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl). In some embodiments, the amino and/oralkyl of the optionally substituted aminoalkyl is substituted with oneor more of optionally substituted alkyl, optionally substituted alkenyl,optionally substituted sulfoalkyl, optionally substituted carboxy (e.g.,substituted with an O-protecting group), optionally substituted hydroxy(e.g., substituted with an O-protecting group), optionally substitutedcarboxyalkyl (e.g., substituted with an O-protecting group), optionallysubstituted alkoxycarbonylalkyl (e.g., substituted with an O-protectinggroup), or N-protecting group. In some embodiments, optionallysubstituted aminoalkyl is substituted with an optionally substitutedsulfoalkyl or optionally substituted alkenyl. In particular embodiments,R^(12a) and R^(Vb″) are both H. In particular embodiments, T¹ is O(oxo), and T² is S (thio) or Se (seleno).

In some embodiments,R^(Vb′ is optionally substituted alkoxycarbonylalkyl or optionally substituted carbamoylalkyl.)

In particular embodiments, the optional substituent for R^(12a),R^(12b), R^(12c), or R^(Va) is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B is a modified cytosine. Exemplary modifiedcytosines include compounds (b10)-(b14):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(3′) and T^(3″) is, independently, H, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substitutedthioalkoxy, or the combination of T^(3′) and T^(3″) join together (e.g.,as in T³) to form O (oxo), S (thio), or Se (seleno);

each V⁴ is, independently, O, S, N(R^(Vc))_(nv), or C(R^(Vc))_(nv),wherein nv is an integer from 0 to 2 and each R^(Vc) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl), wherein the combination of R^(13b)and R^(Vc) can be taken together to form optionally substitutedheterocyclyl;

each V⁵ is, independently, N(R^(Vd))_(nb), or C(R^(Vd))_(nv), wherein nvis an integer from 0 to 2 and each R^(Vd) is, independently, H, halo,optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl) (e.g., V⁵ is —CH or N);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

Further exemplary modified cytosines include those having Formula(b32)-(b35):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T¹ and T³ is, independently, O (oxo), S (thio), or Se (seleno);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl,alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl (e.g.,R¹⁵ is H, and R¹⁶ is H or optionally substituted alkyl).

In some embodiments, R¹⁵ is H, and R¹⁶ is H or optionally substitutedalkyl. In particular embodiments, R¹⁴ is H, acyl, or hydroxyalkyl. Insome embodiments, R¹⁴ is halo. In some embodiments, both R¹⁴ and R¹⁵ areH. In some embodiments, both R¹⁵ and R¹⁶ are H. In some embodiments,each of R¹⁴ and R¹⁵ and R¹⁶ is H. In further embodiments, each ofR^(13a) and R^(13b) is independently, H or optionally substituted alkyl.

Further non-limiting examples of modified cytosines include compounds ofFormula (b36):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each R^(13b) is, independently, H, optionally substituted acyl,optionally substituted acyloxyalkyl, optionally substituted alkyl, oroptionally substituted alkoxy, wherein the combination of R^(13b) andR^(14b) can be taken together to form optionally substitutedheterocyclyl;

each R^(14a) and R^(14b) is, independently, H, halo, hydroxy, thiol,optionally substituted acyl, optionally substituted amino acid,optionally substituted alkyl, optionally substituted haloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted hydroxyalkyl (e.g., substituted with anO-protecting group), optionally substituted hydroxyalkenyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted acyloxyalkyl,optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl,phosphoryl, optionally substituted aminoalkyl, or optionally substitutedcarboxyaminoalkyl), azido, optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl; and

each of R¹⁵ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

In particular embodiments, R^(14b) is an optionally substituted aminoacid (e.g., optionally substituted lysine). In some embodiments, R^(14a)is H.

In some embodiments, B is a modified guanine. Exemplary modifiedguanines include compounds of Formula (b15)-(b17):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(4′), T^(4″), T^(5′), T^(5″), T^(6′), and T^(6″) isindependently, H, optionally substituted alkyl, or optionallysubstituted alkoxy, and wherein the combination of T^(4′) and T^(4″)(e.g., as in T⁴) or the combination of T^(5′) and T^(5″) (e.g., as inT⁵) or the combination of T^(6′) and T^(6″) join together (e.g., as inT⁶) form O (oxo), S (thio), or Se (seleno);

each of V⁵ and V⁶ is, independently, O, S, N(R^(Vd))_(nv), orC(R^(Vd))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vd) is,independently, H, halo, thiol, optionally substituted amino acid, cyano,amidine, optionally substituted aminoalkyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), optionally substitutedthioalkoxy, or optionally substituted amino; and

each of R¹⁷, R¹⁸, R^(19a), R^(19b), R²¹, R²², R²³, and R²⁴ is,independently, H, halo, thiol, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted thioalkoxy, optionally substituted amino, or optionallysubstituted amino acid.

Exemplary modified guanosines include compounds of Formula (b37)-(b40):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(4′) is, independently, H, optionally substituted alkyl, oroptionally substituted alkoxy, and each T⁴ is, independently, O (oxo), S(thio), or Se (seleno);

each of R¹⁸, R^(19a), R^(19b), and R²¹ is, independently, H, halo,thiol, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted thioalkoxy,optionally substituted amino, or optionally substituted amino acid.

In some embodiments, R¹⁸ is H or optionally substituted alkyl. Infurther embodiments, T⁴ is oxo. In some embodiments, each of R^(19a) andR^(19b) is, independently, H or optionally substituted alkyl.

In some embodiments, B is a modified adenine. Exemplary modifiedadenines include compounds of Formula (b18)-(b20):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each V⁷ is, independently, O, S, N(R^(Ve))_(nv), or C(R^(Ve))_(nv),wherein nv is an integer from 0 to 2 and each R^(Ve) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy (e.g., optionally substituted with anysubstituent described herein, such as those selected from (1)-(21) foralkyl);

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl);

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy or optionallysubstituted amino;

each R²⁸ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, or optionally substituted alkynyl; and

each R²⁹ is, independently, H, optionally substituted acyl, optionallysubstituted amino acid, optionally substituted carbamoylalkyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted alkoxy, or optionallysubstituted amino.

Exemplary modified adenines include compounds of Formula (b41)-(b43):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl); and

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy, or optionallysubstituted amino.

In some embodiments, R^(26a) is H, and R^(26b) is optionally substitutedalkyl. In some embodiments, each of R^(26a) and R^(26b) is,independently, optionally substituted alkyl. In particular embodiments,R²⁷ is optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy. In other embodiments, R²⁵ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy.

In particular embodiments, the optional substituent for R^(26a),R^(26b), or R²⁹ is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B may have Formula (b21):

wherein X¹² is, independently, O, S, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene, xa is aninteger from 0 to 3, and R^(12a) and T² are as described herein.

In some embodiments, B may have Formula (b22):

wherein R^(10′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R¹¹, R^(12a), T¹, and T² are asdescribed herein.

In some embodiments, B may have Formula (b23):

wherein R¹⁰ is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substitued thienyl, or optionallysubstitued pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁰); and wherein R¹¹ (e.g., Hor any substituent described herein), R^(12a) (e.g., H or anysubstituent described herein), T¹ (e.g., oxo or any substituentdescribed herein), and T² (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B may have Formula (b24):

wherein R^(14′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted alkaryl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R^(13a), R^(13b), R¹⁵, and T³ are asdescribed herein.

In some embodiments, B may have Formula (b25):

wherein R^(14′) is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substitued thienyl, or optionallysubstitued pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁴ or R^(14′)); and whereinR^(13a) (e.g., H or any substituent described herein), R^(13b) (e.g., Hor any substituent described herein), R¹⁵ (e.g., H or any substituentdescribed herein), and T³ (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B is a nucleobase selected from the groupconsisting of cytosine, guanine, adenine, and uracil. In someembodiments, B may be:

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τcm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine(m⁶A),2-methylthio-N-6-methyl-adenosine (ms²m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N-6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m′Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²′⁷G), N2,N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,a-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m′Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modifications on the Internucleoside Linkage

The modified nucleosides and nucleotides, which may be incorporated intoa modified nucleic acid or mmRNA molecule, can be modified on theinternucleoside linkage (e.g., phosphate backbone). The phosphate groupsof the backbone can be modified by replacing one or more of the oxygenatoms with a different substituent. Further, the modified nucleosidesand nucleotides can include the wholesale replacement of an unmodifiedphosphate moiety with a modified phosphate as described herein. Examplesof modified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenates, boranophosphates, boranophosphateesters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates,alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioateshave both non-linking oxygens replaced by sulfur. The phosphate linkercan also be modified by the replacement of a linking oxygen withnitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates),and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked modified nucleic acids or mmRNAmolecules are expected to also reduce the innate immune response throughweaker binding/activation of cellular innate immune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine,5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The modified nucleic acids and mmRNA of the invention can include acombination of modifications to the sugar, the nucleobase, and/or theinternucleoside linkage. These combinations can include any one or moremodifications described herein. For examples, any of the nucleotidesdescribed herein in Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IVl), and (IXa)-(IXr) can be combined with any of the nucleobasesdescribed herein (e.g., in Formulas (b1)-(b43) or any other describedherein).

Synthesis of Modified Nucleic Acids and mmRNA Molecules

The modified nucleic acid and mmRNA molecules for use in accordance withthe invention may be prepared according to any useful technique, asdescribed herein. The modified nucleosides and nucleotides used in thesynthesis of modified nucleic acid and mmRNA molecules disclosed hereincan be prepared from readily available starting materials using thefollowing general methods and procedures. Where typical or preferredprocess conditions (e.g., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are provided, a skilled artisanwould be able to optimize and develop additional process conditions.Optimum reaction conditions may vary with the particular reactants orsolvent used, but such conditions can be determined by one skilled inthe art by routine optimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of modified nucleic acid and mmRNA molecules of the presentinvention can involve the protection and deprotection of variouschemical groups. The need for protection and deprotection, and theselection of appropriate protecting groups can be readily determined byone skilled in the art. The chemistry of protecting groups can be found,for example, in Greene, et al., Protective Groups in Organic Synthesis,2d. Ed., Wiley & Sons, 1991, which is incorporated herein by referencein its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotidescan be carried out by any of numerous methods known in the art. Anexample method includes fractional recrystallization using a “chiralresolving acid” which is an optically active, salt-forming organic acid.Suitable resolving agents for fractional recrystallization methods are,for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids. Resolution of racemic mixtures can also becarried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Modified nucleosides and nucleotides (e.g., binding block molecules) canbe prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The modified nucleic acid and mmRNA of the invention need not beuniformly modified along the entire length of the molecule. For example,one or more or all types of nucleotide (e.g., purine or pyrimidine, orany one or more or all of A, G, U, C) may or may not be uniformlymodified in a polynucleotide of the invention, or in a givenpredetermined sequence region thereof. In some embodiments, allnucleotides X in a polynucleotide of the invention (or in a givensequence region thereof) are modified, wherein X may any one ofnucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C,G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the modified nucleic acid or mmRNA. One of ordinaryskill in the art will appreciate that the nucleotide analogs or othermodification(s) may be located at any position(s) of a modified nucleicacid or mmRNA such that the function of the modified nucleic acid ormmRNA is not substantially decreased. A modification may also be a 5′ or3′ terminal modification. The modified nucleic acid or mmRNA may containfrom about 1% to about 100% modified nucleotides, or any interveningpercentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%,from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20%to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%).

In some embodiments, the modified nucleic acid or mmRNA includes amodified pyrimidine (e.g., a modified uracil/uridine or modifiedcytosine/cytidine). In some embodiments, the uracil or uridine in themodified nucleic acid or mmRNA molecule may be replaced with from about1% to about 100% of a modified uracil or modified uridine (e.g., from 1%to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%,from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10%to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%,from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%,from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70%to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to95%, from 90% to 100%, and from 95% to 100% of a modified uracil ormodified uridine). The modified uracil or uridine can be replaced by acompound having a single unique structure or by a plurality of compoundshaving different structures (e.g., 2, 3, 4 or more unique structures, asdescribed herein). In some embodiments, the cytosine or cytidine in themodified nucleic acid or mmRNA molecule may be replaced with from about1% to about 100% of a modified cytosine or modified cytidine (e.g., from1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%,from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20%to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%,from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%,from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%,from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modifiedcytosine or modified cytidine). The modified cytosine or cytidine can bereplaced by a compound having a single unique structure or by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures, as described herein).

In some embodiments, the present disclosure provides methods ofsynthesizing a modified nucleic acid or mmRNA including n number oflinked nucleosides having Formula (Ia-1)

comprising:

a) reacting a nucleotide of Formula (IV-1):

with a phosphoramidite compound of Formula (V-1):

wherein Y⁹ is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino,thiol, optionally substituted amino acid, or a peptide (e.g., includingfrom 2 to 12 amino acids); and each P¹, P², and P³ is, independently, asuitable protecting group; and

denotes a solid support;

to provide a modified nucleic acid or mmRNA of Formula (VI-1):

and

b) oxidizing or sulfurizing the modified nucleic acid or mmRNA ofFormula (V) to yield a modified nucleic acid or mmRNA of Formula(VII-1):

and

c) removing the protecting groups to yield the modified nucleic acid ormmRNA of Formula (Ia).

In some embodiments, steps a) and b) are repeated from 1 to about 10,000times. In some embodiments, the methods further comprise a nucleotide(e.g., building block molecule) selected from the group consisting ofadenosine, cytosine, guanosine, and uracil. In some embodiments, thenucleobase may be a pyrimidine or derivative thereof. In someembodiments, the modified nucleic acid or mmRNA is translatable.

Other components of modified nucleic acids and mmRNA are optional, andare beneficial in some embodiments. For example, a 5′ untranslatedregion (UTR) and/or a 3′UTR are provided, wherein either or both mayindependently contain one or more different nucleoside modifications. Insuch embodiments, nucleoside modifications may also be present in thetranslatable region. Also provided are modified nucleic acids and mmRNAcontaining a Kozak sequence.

Exemplary syntheses of modified nucleotides, which are incorporated intoa modified nucleic acid or mmRNA, e.g., RNA or mRNA, are provided belowin Scheme 1 through Scheme 11. Scheme 1 provides a general method forphosphorylation of nucleosides, including modified nucleosides.

Various protecting groups may be used to control the reaction. Forexample, Scheme 2 provides the use of multiple protecting anddeprotecting steps to promote phosphorylation at the 5′ position of thesugar, rather than the 2′ and 3′ hydroxyl groups.

Modified nucleotides can be synthesized in any useful manner. Schemes 3,4, and 7 provide exemplary methods for synthesizing modified nucleotideshaving a modified purine nucleobase; and Schemes 5 and 6 provideexemplary methods for synthesizing modified nucleotides having amodified pseudouridine or pseudoisocytidine, respectively.

Schemes 8 and 9 provide exemplary syntheses of modified nucleotides.Scheme 10 provides a non-limiting biocatalytic method for producingnucleotides.

Scheme 11 provides an exemplary synthesis of a modified uracil, wherethe N1 position is modified with R^(12b), as provided elsewhere, and the5′-position of ribose is phosphorylated. T¹, T², R^(12a), R^(12b), and rare as provided herein. This synthesis, as well as optimized versionsthereof, can be used to modify other pyrimidine nucleobases and purinenucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one ormore phosphate groups (e.g., at the 5′ position of the sugar). Thisalkylating reaction can also be used to include one or more optionallysubstituted alkyl group at any reactive group (e.g., amino group) in anynucleobase described herein (e.g., the amino groups in the Watson-Crickbase-pairing face for cytosine, uracil, adenine, and guanine).

Combinations of Nucleotides in mmRNA

Further examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 2. These combinations ofmodified nucleotides can be used to form the modified nucleic acids ormmRNA of the invention. Unless otherwise noted, the modified nucleotidesmay be completely substituted for the natural nucleotides of themodified nucleic acids or mmRNA of the invention. As a non-limitingexample, the natural nucleotide uridine may be substituted with amodified nucleoside described herein. In another non-limiting example,the natural nucleotide uridine may be partially substituted (e.g., about0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of themodified nucleoside disclosed herein.

TABLE 2 Modified Nucleotide Modified Nucleotide Combinationα-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudo-uridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about50% of the cytosines are α-thio-cytidine pseudoisocytidinepseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines areN1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1-methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/about 50%of uridines are 2-thio-uridine N4-acetyl-N4-acetyl-cytidine/5-iodo-uridine cytidineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/about 50% of uridines are 2-thio-uridine

Further examples of modified nucleotide combinations are provided belowin Table 3. These combinations of modified nucleotides can be used toform the modified nucleic acid molecules or mmRNA of the invention.

TABLE 3 Modified Nucleotide Modified Nucleotide Combination modifiedcytidine modified cytidine with (b10)/pseudouridine having one ormodified cytidine with (b10)/N1-methyl-pseudouridine more nucleobasesmodified cytidine with (b10)/5-methoxy-uridine of Formula (b10) modifiedcytidine with (b10)/5-methyl-uridine modified cytidine with(b10)/5-bromo-uridine modified cytidine with (b10)/2-thio-uridine about50% of cytidine substituted with modified cytidine (b10)/about 50% ofuridines are 2-thio-uridine modified cytidine modified cytidine with(b32)/pseudouridine having one or modified cytidine with(b32)/N1-methyl-pseudouridine more nucleobases modified cytidine with(b32)/5-methoxy-uridine of Formula (b32) modified cytidine with(b32)/5-methyl-uridine modified cytidine with (b32)/5-bromo-uridinemodified cytidine with (b32)/2-thio-uridine about 50% of cytidinesubstituted with modified cytidine (b32)/about 50% of uridines are2-thio-uridine modified uridine modified uridine with(b1)/N4-acetyl-cytidine having one or modified uridine with(b1)/5-methyl-cytidine more nucleobases of Formula (b1) modified uridinemodified uridine with (b8)/N4-acetyl-cytidine having one or modifieduridine with (b8)/5-methyl-cytidine more nucleobases of Formula (b8)modified uridine modified uridine with (b28)/N4-acetyl-cytidine havingone or modified uridine with (b28)/5-methyl-cytidine more nucleobases ofFormula (b28) modified uridine modified uridine with(b29)/N4-acetyl-cytidine having one or modified uridine with(b29)/5-methyl-cytidine more nucleobases of Formula (b29) modifieduridine modified uridine with (b30)/N4-acetyl-cytidine having one ormodified uridine with (b30)/5-methyl-cytidine more nucleobases ofFormula (b30)

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14) (e.g., at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9) (e.g., at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), and at least 25% of the uracils arereplaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

Synthesis of Modified Nucleic Acid Molecules

Modified nucleic acid molecules for use in accordance with the presentdisclosure may be prepared according to any available techniqueincluding, but not limited to, in vitro transcription such as chemicalsynthesis and enzymatic synthesis, or enzymatic and chemical cleavage ofa longer precursor, etc. Methods of synthesizing RNA are known in theart (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practicalapproach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; andHerdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications,Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.:Humana Press, 2005; both of which are incorporated herein by reference).

The modified nucleic acid molecules disclosed herein can be preparedfrom readily available starting materials using the following generalmethods and procedures. It is understood that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given other process conditionscan also be used unless otherwise stated. Optimum reaction conditionsmay vary with the particular reactants or solvent used, but suchconditions can be determined by one skilled in the art by routineoptimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), mass spectrometry, or by chromatography such as highperformance liquid chromatography (HPLC) or thin layer chromatography.

Preparation of modified nucleic acid molecules can involve theprotection and deprotection of various chemical groups. The need forprotection and deprotection, and the selection of appropriate protectinggroups can be readily determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Greene, etal., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleic acid molecules can becarried out by any of numerous methods known in the art. An examplemethod includes, but is not limited to, fractional recrystallizationusing a “chiral resolving acid” which is an optically active,salt-forming organic acid. Suitable resolving agents for fractionalrecrystallization methods are, for example, optically active acids, suchas the D and L forms of tartaric acid, diacetyltartaric acid,dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or thevarious optically active camphorsulfonic acids. Resolution of racemicmixtures can also be carried out by elution on a column packed with anoptically active resolving agent (e.g., dinitrobenzoylphenylglycine).Suitable elution solvent composition can be determined by one skilled inthe art.

Modified nucleic acid molecules need not be uniformly modified along theentire length of the molecule. Different nucleic acid modificationsand/or backbone structures may exist at various positions in the nucleicacid. One of ordinary skill in the art will appreciate that thenucleotide analogs or other modification(s) may be located at anyposition(s) of a nucleic acid such that the function of the nucleic acidis not substantially decreased. A modification may also be a 5′ or 3′terminal modification. The nucleic acids may contain at a minimum onemodified nucleotide and at maximum 100% modified nucleotides, or anyintervening percentage, such as at least 5% modified nucleotides, atleast 10% modified nucleotides, at least 25% modified nucleotides, atleast 50% modified nucleotides, at least 80% modified nucleotides, or atleast 90% modified nucleotides. For example, the nucleic acids maycontain a modified pyrimidine such as uracil or cytosine. In someembodiments, at least 5%, at least 10%, at least 25%, at least 50%, atleast 80%, at least 90% or 100% of the uracil in the nucleic acid may bereplaced with a modified uracil. The modified uracil can be replaced bya compound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures). In some embodiments, at least 5%, at least 10%,at least 25%, at least 50%, at least 80%, at least 90% or 100% of thecytosine in the nucleic acid may be replaced with a modified cytosine.The modified cytosine can be replaced by a compound having a singleunique structure, or can be replaced by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures).

Generally, the shortest length of a modified mRNA, herein “mmRNA,” ofthe present disclosure can be the length of an mRNA sequence that may besufficient to encode for a dipeptide. In another embodiment, the lengthof the mRNA sequence may be sufficient to encode for a tripeptide. Inanother embodiment, the length of an mRNA sequence may be sufficient toencode for a tetrapeptide. In another embodiment, the length of an mRNAsequence may be sufficient to encode for a pentapeptide. In anotherembodiment, the length of an mRNA sequence may be sufficient to encodefor a hexapeptide. In another embodiment, the length of an mRNA sequencemay be sufficient to encode for a heptapeptide. In another embodiment,the length of an mRNA sequence may be sufficient to encode for anoctapeptide. In another embodiment, the length of an mRNA sequence maybe sufficient to encode for a nonapeptide. In another embodiment, thelength of an mRNA sequence may be sufficient to encode for adecapeptide.

Examples of dipeptides that the modified nucleic acid molecule sequencescan encode for include, but are not limited to, carnosine and anserine.

In a further embodiment, the mRNA may be greater than 30 nucleotides inlength. In another embodiment, the RNA molecule may be greater than 35nucleotides in length (e.g., at least or greater than about 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400,1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000 and 5,000nucleotides).

Exemplary Properties of Modified Nucleic Acid Molecules

Major Groove Interacting Partners

The modified nucleic acid molecules, e.g., modified mRNA (mmRNA),described herein can disrupt interactions with recognition receptorsthat detect and respond to RNA ligands through interactions, e.g.binding, with the major groove face of a nucleotide or nucleic acid. Assuch, RNA ligands comprising modified nucleotides or modified nucleicacid molecules, as described herein, decrease interactions with majorgroove binding partners, and therefore decrease an innate immuneresponse, or expression and secretion of pro-inflammatory cytokines, orboth.

Example major groove interacting, e.g. binding, partners include, butare not limited to, the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNA. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNA toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MDA5 (melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

Prevention or Reduction of Innate Cellular Immune Response ActivationUsing Modified Nucleic Acid Molecules

The modified nucleic acid molecules, e.g., mmRNA, described herein,decrease the innate immune response in a cell. The term “innate immuneresponse” includes a cellular response to exogenous nucleic acids,including, but not limited to, single stranded nucleic acids, generallyof viral or bacterial origin, which involve the induction of cytokineexpression and release, particularly the interferons, and cell death.Protein synthesis may also be reduced during the innate cellular immuneresponse. While it is advantageous to eliminate the innate immuneresponse in a cell, the present disclosure provides modified mRNA thatsubstantially reduce the immune response, including interferonsignaling, without entirely eliminating such a response. In someembodiments, the immune response may be reduced by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% ascompared to the immune response induced by a corresponding unmodifiednucleic acid molecule. Such a reduction can be measured by theexpression or activity level of Type 1 interferons or the expression ofinterferon-regulated genes such as the toll-like receptors (e.g., TLR7and TLR8). Reduction of the innate immune response can also be measuredby decreased cell death following one or more administrations ofmodified RNA to a cell population; e.g., cell death is 10%, 25%, 50%,75%, 85%, 90%, 95%, or over 95% less than the cell death frequencyobserved with a corresponding unmodified nucleic acid molecule.Moreover, cell death may affect fewer than 50%, 40%, 30%, 20%, 10%, 5%,1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the modifiednucleic acid molecules.

The present disclosure provides for the repeated introduction (e.g.,transfection) of modified nucleic acid molecules into a target cellpopulation, e.g., in vitro, ex vivo, or in vivo. The step of contactingthe cell population may be repeated one or more times (such as two,three, four, five or more than five times). In some embodiments, thestep of contacting the cell population with the modified nucleic acidmolecules may be repeated a number of times sufficient such that apredetermined efficiency of protein translation in the cell populationis achieved. Given the reduced cytotoxicity of the target cellpopulation by the nucleic acid modifications, such repeatedtransfections are achievable in a variety of cell types.

The modified nucleic acids of the invention, including the combinationof modifications taught herein may have superior properties making themmore suitable as therapeutic modalities.

It has been determined that the “all or none” model in the art is sorelyinsufficient to describe the biological phenomena associated with thetherapeutic utility of modified mRNA. The present inventors havedetermined that to improve protein production, one may consider thenature of the modification, or combination of modifications, the percentmodification and survey more than one cytokine or metric to determinethe efficacy and risk profile of a particular modified mRNA.

In one aspect of the invention, methods of determining the effectivenessof a modified mRNA as compared to unmodified involves the measure andanalysis of one or more cytokines whose expression is triggered by theadministration of the exogenous nucleic acid of the invention. Thesevalues are compared to administration of an umodified nucleic acid or toa standard metric such as cytokine response, PolyIC, R-848 or otherstandard known in the art.

One example of a standard metric developed herein is the measure of theratio of the level or amount of encoded polypeptide (protein) producedin the cell, tissue or organism to the level or amount of one or more(or a panel) of cytokines whose expression is triggered in the cell,tissue or organism as a result of administration or contact with themodified nucleic acid. Such ratios are referred to herein as theProtein:Cytokine Ratio or “PC” Ratio. The higher the PC ratio, the moreefficacioius the modified nucleic acid (polynucleotide encoding theprotein measured). Preferred PC Ratios, by cytokine, of the presentinvention may be greater than 1, greater than 10, greater than 100,greater than 1000, greater than 10,000 or more. Modified nucleic acidshaving higher PC Ratios than a modified nucleic acid of a different orunmodified construct are preferred.

The PC ratio may be further qualified by the percent modificationpresent in the polynucleotide. For example, normalized to a 100%modified nucleic acid, the protein production as a function of cytokine(or risk) or cytokine profile can be determined.

In one embodiment, the present invention provides a method fordetermining, across chemistries, cytokines or percent modification, therelative efficacy of any particular modified polynucleotide by comparingthe PC Ratio of the modified nucleic acid (polynucleotide).

Activation of the Immune Response: Vaccines

In one embodiment of the present invention, mRNA molecules may be usedto elicit or provoke an immune response in an organism. The mRNAmolecules to be delivered may encode an immunogenic peptide orpolypeptide and may encode more than one such peptide or polypeptide.

Additionally, certain modified nucleosides, or combinations thereof,when introduced into the modified nucleic acid molecules or mmRNA of theinvention will activate the innate immune response. Such activatingmolecules are useful as adjuvants when combined with polypeptides and/orother vaccines. In certain embodiments, the activating molecules containa translatable region which encodes for a polypeptide sequence useful asa vaccine, thus providing the ability to be a self-adjuvant.

In one embodiment, the modified nucleic acid molecules and/or mmRNA ofthe invention may encode an immunogen. The delivery of modified nucleicacid molecules and/or mmRNA encoding an immunogen may activate theimmune response. As a non-limiting example, the modified nucleic acidmolecules and/or mmRNA encoding an immunogen may be delivered to cellsto trigger multiple innate response pathways (see International Pub. No.WO2012006377; herein incorporated by reference in its entirety). Asanother non-limiting example, the modified nucleic acid molecules andmmRNA of the present invention encoding an immunogen may be delivered toa vertebrate in a dose amount large enough to be immunogenic to thevertebrate (see International Pub. No. WO2012006372 and WO2012006369;each of which is herein incorporated by reference in their entirety).

The modified nucleic acid molecules or mmRNA of invention may encode apolypeptide sequence for a vaccine and may further comprise aninhibitor. The inhibitor may impair antigen presentation and/or inhibitvarious pathways known in the art. As a non-limiting example, themodified nucleic acid molecules or mmRNA of the invention may be usedfor a vaccine in combination with an inhibitor which can impair antigenpresentation (see International Pub. No. WO2012089225 and WO2012089338;each of which is herein incorporated by reference in their entirety).

In one embodiment, the modified nucleic acid molecules or mmRNA of theinvention may be self-replicating RNA. Self-replicating RNA moleculescan enhance efficiency of RNA delivery and expression of the enclosedgene product. In one embodiment, the modified nucleic acid molecules ormmRNA may comprise at least one modification described herein and/orknown in the art. In one embodiment, the self-replicating RNA can bedesigned so that the self-replicating RNA does not induce production ofinfectious viral particles. As a non-limiting example theself-replicating RNA may be designed by the methods described in US Pub.No. US20110300205 and International Pub. No. WO2011005799, each of whichis herein incorporated by reference in their entirety.

In one embodiment, the self-replicating modified nucleic acid moleculesor mmRNA of the invention may encode a protein which may raise theimmune response. As a non-limiting example, the modified nucleic acidmolecules and/or mmRNA may be self-replicating mRNA may encode at leastone antigen (see US Pub. No. US20110300205 and International Pub. No.WO2011005799; each of which is herein incorporated by reference in theirentirety).

In one embodiment, the self-replicating modified nucleic acids or mmRNAof the invention may be formulated using methods described herein orknown in the art. As a non-limiting example, the self-replicating RNAmay be formulated for delivery by the methods described in Geall et al(Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID:22908294; herein incorporated by reference in its entirety).

In one embodiment, the modified nucleic acid molecules or mmRNA of thepresent invention may encode amphipathic and/or immunogenic amphipathicpeptides.

In on embodiment, a formulation of the modified nucleic acid moleculesor mmRNA of the present invention may further comprise an amphipathicand/or immunogenic amphipathic peptide. As a non-limiting example, themodified nucleic acid molecule or mmRNA comprising an amphipathic and/orimmunogenic amphipathic peptide may be formulated as described in US.Pub. No. US20110250237 and International Pub. Nos. WO2010009277 andWO2010009065; each of which is herein incorporated by reference in theirentirety.

In one embodiment, the modified nucleic acid molecules and mmRNA of thepresent invention may be immunostimultory. As a non-limiting example,the modified nucleic acid molecules and mmRNA may encode all or a partof a positive-sense or a negative-sense stranded RNA virus genome (seeInternational Pub No. WO2012092569 and US Pub No. US20120177701, each ofwhich is herein incorporated by reference in their entirety). In anothernon-limiting example, the immunostimultory modified nucleic acidmolecules or mmRNA of the present invention may be formulated with anexcipient for administration as described herein and/or known in the art(see International Pub No. WO2012068295 and US Pub No. US20120213812,each of which is herein incorporated by reference in their entirety).

In one embodiment, the response of the vaccine formulated by the methodsdescribed herein may be enhanced by the addition of various compounds toinduce the therapeutic effect. As a non-limiting example, the vaccineformulation may include a MHC II binding peptide or a peptide having asimilar sequence to a MHC II binding peptide (see International Pub Nos.WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965,each of which is herein incorporated by reference in their entirety). Asanother example, the vaccine formulations may comprise modifiednicotinic compounds which may generate an antibody response to nicotineresidue in a subject (see International Pub No. WO2012061717 and US PubNo. US20120114677, each of which is herein incorporated by reference intheir entirety).

Polypeptide Variants

The modified nucleic acid molecules encode polypeptides, e.g., a variantpolypeptides, which have a certain identity to a reference polypeptidesequence. The term “identity,” as known in the art, refers to arelationship between the sequences of two or more peptides, determinedby comparing the sequences. In the art, “identity” also refers to thedegree of sequence relatedness between peptides, as determined by thenumber of matches between strings of two or more amino acid residues.Identity measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related peptides can be readily calculated by known methods.Such methods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988); all of which are hereinincorporated by reference in their entirety.

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the present disclosure will have atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of this present disclosure. For example, providedherein is any protein fragment of a reference protein (meaning apolypeptide sequence which is at least one amino acid residue shorterthan a reference polypeptide sequence but otherwise identical) 10, 20,30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the present disclosure. In certainembodiments, a protein sequence to be utilized in accordance with thepresent disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or moremutations as shown in any of the sequences provided or referencedherein.

Polypeptide-Nucleic Acid Complexes

Proper protein translation involves the physical aggregation of a numberof polypeptides and nucleic acids associated with the mRNA. Provided bythe present disclosure are protein-nucleic acid complexes, containing atranslatable mRNA having one or more nucleoside modifications (e.g., atleast two different nucleoside modifications) and one or morepolypeptides bound to the mRNA. Generally, the proteins are provided inan amount effective to prevent or to reduce an innate immune response ofa cell into which the complex is introduced.

Untranslatable Modified Nucleic Acid Molecules

As described herein, provided are mRNA having sequences that aresubstantially not translatable. Such mRNA may be effective as a vaccinewhen administered to a subject. It is further provided that the subjectadministered the vaccine may be a mammal, more preferably a human andmost preferably a patient.

Also provided are modified nucleic acid molecules that contain one ormore noncoding regions. Such modified nucleic acid molecules aregenerally not translated, but are capable of binding to and sequesteringone or more translational machinery component such as a ribosomalprotein or a transfer RNA (tRNA), thereby effectively reducing theprotein expression in the cell. The modified nucleic acid molecule maycontain a small nucleolar RNA (sno-RNA), micro RNA (miRNA), smallinterfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

Pharmaceutical Compositions Formulation, Administration, Delivery andDosing

The present invention provides modified nucleic acids and mmRNAcompositions and complexes in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference in its entirety).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to modified nucleic acidsand mmRNA to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The modified nucleic acid, and mmRNA of the invention can be formulatedusing one or more excipients to: (1) increase stability; (2) increasecell transfection; (3) permit the sustained or delayed release (e.g.,from a depot formulation of the modified nucleic acid, or mmRNA); (4)alter the biodistribution (e.g., target the modified nucleic acid, ormmRNA to specific tissues or cell types); (5) increase the translationof encoded protein in vivo; and/or (6) alter the release profile ofencoded protein in vivo. In addition to traditional excipients such asany and all solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, excipients ofthe present invention can include, without limitation, lipidoids,liposomes, lipid nanoparticles, polymers, lipoplexes, core-shellnanoparticles, peptides, proteins, cells transfected with modifiednucleic acid, or mmRNA (e.g., for transplantation into a subject),hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the invention can include one or moreexcipients, each in an amount that together increases the stability ofthe modified nucleic acid, or mmRNA, increases cell transfection by themodified nucleic acid, or mmRNA, increases the expression of modifiednucleic acid, or mmRNA encoded protein, and/or alters the releaseprofile of modified nucleic acid, or mmRNA encoded proteins. Further,the modified nucleic acids and mmRNA of the present invention may beformulated using self-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the modified mRNA formulations described herein maycontain at least one modified mRNA. The formulations may contain 1, 2,3, 4 or 5 modified mRNA. In one embodiment, the formulation contains atleast three modified mRNA encoding proteins. In one embodiment, theformulation contains at least five modified mRNA encoding proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference in its entirety). The use of a conventionalexcipient medium may be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium may beincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of modified nucleic acid molecules or mmRNA (see Mahon et al.,Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010267:9-21; Akinc et al., Nat. Biotechnol. 2008 26:561-569; Love et al.,Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc NatlAcad Sci USA. 2011 108:12996-3001; all of which are incorporated hereinin their entireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat. Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol. Ther. 2009 17:872-879; Love et al., Proc Natl AcadSci USA. 2010 107:1864-1869; Leuschner et al., Nat. Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded modified nucleic acid molecules or mmRNA. Complexes,micelles, liposomes or particles can be prepared containing theselipidoids and therefore, can result in an effective delivery of themodified nucleic acid molecules or mmRNA, as judged by the production ofan encoded protein, following the injection of a lipidoid formulationvia localized and/or systemic routes of administration. Lipidoidcomplexes of modified nucleic acid molecules or mmRNA can beadministered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as, but not limited to, particle size(Akinc et al., Mol. Ther. 2009 17:872-879; herein incorporated byreference in its entirety). As an example, small changes in the anchorchain length of poly(ethylene glycol) (PEG) lipids may result insignificant effects on in vivo efficacy. Formulations with the differentlipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010); herein incorporated by reference in its entirety),C12-200 (including derivatives and variants), and MD1, can be tested forin vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol. Ther. 2009 17:872-879 and is incorporated by reference in itsentirety (See FIG. 1).

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670 (see FIG. 1); both of which are hereinincorporated by reference in their entirety. The lipidoid formulationscan include particles comprising either 3 or 4 or more components inaddition to modified nucleic acid molecules or mmRNA. As an example,formulations with certain lipidoids, include, but are not limited to,98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14alkyl chain length). As another example, formulations with certainlipidoids, include, but are not limited to, C12-200 and may contain 50%lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5%PEG-DMG.

In one embodiment, a modified nucleic acid molecule or mmRNA formulatedwith a lipidoid for systemic intravenous administration can target theliver. For example, a final optimized intravenous formulation usingmodified nucleic acid molecule or mmRNA, and comprising a lipid molarcomposition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid with afinal weight ratio of about 7.5 to 1 total lipid to modified nucleicacid, or mmRNA, and a C14 alkyl chain length on the PEG lipid, with amean particle size of roughly 50-60 nm, can result in the distributionof the formulation to be greater than 90% to the liver. (see, Akinc etal., Mol. Ther. 2009 17:872-879; herein incorporated by reference in itsentirety). In another example, an intravenous formulation using aC12-200 (see U.S. provisional application 61/175,770 and publishedinternational application WO2010129709, each of which is hereinincorporated by reference in their entirety) lipidoid may have a molarratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipidto modified nucleic acid molecule or mmRNA, and a mean particle size of80 nm may be effective to deliver modified nucleic acid molecule ormmRNA to hepatocytes (see, Love et al., Proc Natl Acad Sci USA. 2010107:1864-1869 herein incorporated by reference in its entirety). Inanother embodiment, an MD1 lipidoid-containing formulation may be usedto effectively deliver modified nucleic acid molecule or mmRNA tohepatocytes in vivo. The characteristics of optimized lipidoidformulations for intramuscular or subcutaneous routes may varysignificantly depending on the target cell type and the ability offormulations to diffuse through the extracellular matrix into the bloodstream. While a particle size of less than 150 nm may be desired foreffective hepatocyte delivery due to the size of the endothelialfenestrae (see, Akinc et al., Mol. Ther. 2009 17:872-879 hereinincorporated by reference in its entirety), use of a lipidoid-formulatedmodified nucleic acid molecules or mmRNA to deliver the formulation toother cells types including, but not limited to, endothelial cells,myeloid cells, and muscle cells may not be similarly size-limited. Useof lipidoid formulations to deliver siRNA in vivo to othernon-hepatocyte cells such as myeloid cells and endothelium has beenreported (see Akinc et al., Nat. Biotechnol. 2008 26:561-569; Leuschneret al., Nat. Biotechnol. 2011 29:1005-1010; Cho et al. Adv. Funct.Mater. 2009 19:3112-3118; 8^(th) International Judah Folkman Conference,Cambridge, Mass. Oct. 8-9, 2010; each of which is herein incorporated byreference in its entirety). Effective delivery to myeloid cells, such asmonocytes, lipidoid formulations may have a similar component molarratio. Different ratios of lipidoids and other components including, butnot limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG,may be used to optimize the formulation of the modified nucleic acid, ormmRNA for delivery to different cell types including, but not limitedto, hepatocytes, myeloid cells, muscle cells, etc. For example, thecomponent molar ratio may include, but is not limited to, 50% C12-200,10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG(see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; hereinincorporated by reference in its entirety). The use of lipidoidformulations for the localized delivery of nucleic acids to cells (suchas, but not limited to, adipose cells and muscle cells) via eithersubcutaneous or intramuscular delivery, may not require all of theformulation components desired for systemic delivery, and as such maycomprise only the lipidoid and the modified nucleic acid molecule ormmRNA.

Combinations of different lipidoids may be used to improve the efficacyof modified nucleic acid molecule or mmRNA directed protein productionas the lipidoids may be able to increase cell transfection by themodified nucleic acid molecule or mmRNA; and/or increase the translationof encoded protein (see Whitehead et al., Mol. Ther. 2011, 19:1688-1694,herein incorporated by reference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

The modified nucleic acid molecules and mmRNA of the invention can beformulated using one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, pharmaceutical compositions ofmodified nucleic acid molecule or mmRNA include liposomes. Liposomes areartificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). Inone embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat. Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties.) The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the modified nucleicacid molecule or mmRNA. As an example a liposome can contain, but is notlimited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC),10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),as described by Jeffs et al. As another example, certain liposomeformulations may contain, but are not limited to, 48% cholesterol, 20%DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid canbe 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In one embodiment, pharmaceutical compositions may include liposomeswhich may be formed to deliver mmRNA which may encode at least oneimmunogen. The mmRNA may be encapsulated by the liposome and/or it maybe contained in an aqueous core which may then be encapsulated by theliposome (see International Pub. Nos. WO2012031046, WO2012031043,WO2012030901 and WO2012006378; each of which is herein incorporated byreference in their entirety). In another embodiment, the mmRNA which mayencode an immunogen may be formulated in a cationic oil-in-wateremulsion where the emulsion particle comprises an oil core and acationic lipid which can interact with the mmRNA anchoring the moleculeto the emulsion particle (see International Pub. No. WO2012006380;herein incorporated by reference in its entirety). In yet anotherembodiment, the lipid formulation may include at least cationic lipid, alipid which may enhance transfection and a least one lipid whichcontains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; eachof which is herein incorporated by reference in their entirety). Inanother embodiment, the modified mRNA encoding an immunogen may beformulated in a lipid vesicle which may have crosslinks betweenfunctionalized lipid bilayers (see U.S. Pub. No. 20120177724, hereinincorporated by reference in its entirety).

In one embodiment, the modified mRNA may be formulated in a lipidvesicle which may have crosslinks between functionalized lipid bilayers.

In one embodiment, the modified mRNA may be formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished by methods known in the art and/or as described inU.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012013326; herein incorporated byreference in its entirety. In another embodiment, the modified mRNA maybe formulated in a lipid-polycation complex which may further include aneutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol. Ther. 201119:2186-2200; herein incorporated by reference in its entirety).

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain 1-5% of the lipidmolar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC andcholesterol. In another embodiment the PEG-c-DOMG may be replaced with aPEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865 and WO2008103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and8,283,333 and US Patent Publication No. US20100036115 and US20120202871;each of which is herein incorporated by reference in their entirety. Inanother embodiment, the cationic lipid may be selected from, but notlimited to, formula A described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of whichis herein incorporated by reference in their entirety. In yet anotherembodiment, the cationic lipid may be selected from, but not limited to,formula CLI-CLXXIX of International Publication No. WO2008103276,formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII ofU.S. Pat. No. 7,404,969 and formula I-VI of US Patent Publication No.US20100036115; each of which is herein incorporated by reference intheir entirety. As a non-limiting example, the cationic lipid may beselected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimethylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine,(15Z)—N,N-dimethyleptacos-15-en-10-amine,(14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-1 0-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcycIopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 andWO201021865; each of which is herein incorporated by reference in theirentirety.

In one embodiment, the LNP formulation may contain PEG-c-DOMG at 3%lipid molar ratio. In another embodiment, the LNP formulation maycontain PEG-c-DOMG at 1.5% lipid molar ratio.

In one embodiment, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see e.g. Geall et al., Nonviral delivery ofself-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; hereinincorporated by reference in its entirety).

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, each of which is herein incorporated by reference in theirentirety. As a non-limiting example, modified RNA described herein maybe encapsulated in LNP formulations as described in WO2011127255 and/orWO2008103276; each of which is herein incorporated by reference in theirentirety. As another non-limiting example, modified RNA described hereinmay be formulated in a nanoparticle to be delivered by a parenteralroute as described in U.S. Pub. No. 20120207845; herein incorporated byreference in its entirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety. Inanother embodiment, the LNP formulations comprising a polycationiccomposition may be used for the delivery of the modified RNA describedherein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713); herein incorporated by reference in itsentirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a modified nucleic acid molecule(e.g., mmRNA). As a non-limiting example, the carbohydrate carrier mayinclude, but is not limited to, an anhydride-modified phytoglycogen orglycogen-type material, phtoglycogen octenyl succinate, phytoglycogenbeta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g.,International Publication No. WO2012109121; herein incorporated byreference in its entirety).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon. Non-limiting examples of reLNPs include,

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA described herein. In oneembodiment, the lipid nanoparticle may be formulated for use in avaccine such as, but not limited to, against a pathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670, herein incorporated byreference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. The polymeric material mayadditionally be irradiated. As a non-limiting example, the polymericmaterial may be gamma irradiated (See e.g., International App. No.WO201282165, herein incorporated by reference in its entirety).Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer, and(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see e.g., US Publication 20120121718 and USPublication 20100003337 and U.S. Pat. No. 8,263,665; each of which isherein incorporated by reference in their entirety). The co-polymer maybe a polymer that is generally regarded as safe (GRAS) and the formationof the lipid nanoparticle may be in such a way that no new chemicalentities are created. For example, the lipid nanoparticle may comprisepoloxamers coating PLGA nanoparticles without forming new chemicalentities which are still able to rapidly penetrate human mucus (Yang etal. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated byreference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic proteins(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosinβ4 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see e.g., US Publication 20100215580 and US Publication 20080166414;each of which is herein incorporated by reference in their entirety).

The mucus penetrating lipid nanoparticles may comprise at least onemmRNA described herein. The mmRNA may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the particle. The mmRNAmay be covalently coupled to the lipid nanoparticle. Formulations ofmucus penetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In one embodiment, the modified nucleic acid molecule or mmRNA isformulated as a lipoplex, such as, without limitation, the ATUPLEX™system, the DACC system, the DBTC system and other siRNA-lipoplextechnology from Silence Therapeutics (London, United Kingdom), STEMFECT™from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) orprotamine-based targeted and non-targeted delivery of nucleic acidsacids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. IntJ Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 200613:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier etal., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. MicrovascRes 2010 80:286-293 Weide et al. J. Immunother. 2009 32:498-507; Weideet al. J. Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song etal., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl AcadSci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 200819:125-132; all of which are incorporated herein by reference in itsentirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol. Ther. 201018:1357-1364; Song et al., Nat. Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations which have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol. Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit. Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol. Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol. Biol. 2012820:105-116; Ben-Arie et al., Methods Mol. Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol. Biol. 2011 721:339-353;Subramanya et al., Mol. Ther. 2010 18:2028-2037; Song et al., Nat.Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which areincorporated herein by reference in its entirety).

In one embodiment, the modified nucleic acid molecules or mmRNA areformulated as a solid lipid nanoparticle. A solid lipid nanoparticle(SLN) may be spherical with an average diameter between 10 to 1000 nm.SLN possess a solid lipid core matrix that can solubilize lipophilicmolecules and may be stabilized with surfactants and/or emulsifiers. Ina further embodiment, the lipid nanoparticle may be a self-assemblylipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp1696-1702; herein incorporated by reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of modified nucleic acid molecules or mmRNA directed proteinproduction as these formulations may be able to increase celltransfection by the modified nucleic acid molecule or mmRNA; and/orincrease the translation of encoded protein. One such example involvesthe use of lipid encapsulation to enable the effective systemic deliveryof polyplex plasmid DNA (Heyes et al., Mol. Ther. 2007 15:713-720;herein incorporated by reference in its entirety). The liposomes,lipoplexes, or lipid nanoparticles may also be used to increase thestability of the modified nucleic acid molecules or mmRNA.

In one embodiment, the modified nucleic acid molecules and/or the mmRNAof the present invention can be formulated for controlled release and/ortargeted delivery. As used herein, “controlled release” refers to apharmaceutical composition or compound release profile that conforms toa particular pattern of release to effect a therapeutic outcome. In oneembodiment, the modified nucleic acids molecules or the mmRNA may beencapsulated into a delivery agent described herein and/or known in theart for controlled release and/or targeted delivery. As used herein, theterm “encapsulate” means to enclose, surround or encase. As it relatesto the formulation of the compounds of the invention, encapsulation maybe substantial, complete or partial. The term “substantiallyencapsulated” means that at least greater than 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of thepharmaceutical composition or compound of the invention may be enclosed,surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the invention may be enclosed,surrounded or encased within the delivery agent. Advantageously,encapsulation may be determined by measuring the escape or the activityof the pharmaceutical composition or compound of the invention usingfluorescence and/or electron micrograph. For example, at least 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 orgreater than 99.99% of the pharmaceutical composition or compound of theinvention are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106; each of which isherein incorporated by reference in its entirety).

In another embodiment, the modified nucleic acid molecules or the mmRNAmay be encapsulated into a lipid nanoparticle or a rapidly eliminatedlipid nanoparticle and the lipid nanoparticles or a rapidly eliminatedlipid nanoparticle may then be encapsulated into a polymer, hydrogeland/or surgical sealant described herein and/or known in the art. As anon-limiting example, the polymer, hydrogel or surgical sealant may bePLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE®(Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics,San Diego Calif.), surgical sealants such as fibrinogen polymers(Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, IncDeerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International,Inc Deerfield, Ill.).

In another embodiment, the lipid nanoparticle may be encapsulated intoany polymer known in the art which may form a gel when injected into asubject. As a non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the modified nucleic acid molecules or mmRNAformulation for controlled release and/or targeted delivery may alsoinclude at least one controlled release coating. Controlled releasecoatings include, but are not limited to, OPADRY®,polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the controlled release and/or targeted deliveryformulation may comprise at least one degradable polyester which maycontain polycationic side chains. Degradeable polyesters include, butare not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the modified nucleic acid molecules and/or the mmRNAof the present invention may be encapsulated in a therapeuticnanoparticle. Therapeutic nanoparticles may be formulated by methodsdescribed herein and known in the art such as, but not limited to,International Pub Nos. WO2010005740, WO2010030763, WO2010005721,WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286 andUS20120288541, and U.S. Pat. Nos. 8,206,747, 8,293,276 8,318,208 and8,318,211; each of which is herein incorporated by reference in theirentirety. In another embodiment, therapeutic polymer nanoparticles maybe identified by the methods described in US Pub No. US20120140790,herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle may be formulated forsustained release. As used herein, “sustained release” refers to apharmaceutical composition or compound that conforms to a release rateover a specific period of time. The period of time may include, but isnot limited to, hours, days, weeks, months and years. As a non-limitingexample, the sustained release nanoparticle may comprise a polymer and atherapeutic agent such as, but not limited to, the modified nucleic acidmolecules and mmRNA of the present invention (see International Pub No.2010075072 and US Pub No. US20100216804, US20110217377 andUS20120201859, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the therapeutic nanoparticles may be formulated to betarget specific. As a non-limiting example, the thereapeuticnanoparticles may include a corticosteroid (see International Pub. No.WO2011084518 herein incorporated by reference in its entirety). In oneembodiment, the therapeutic nanoparticles of the present invention maybe formulated to be cancer specific. As a non-limiting example, thetherapeutic nanoparticles may be formulated in nanoparticles describedin International Pub No. WO2008121949, WO2010005726, WO2010005725,WO2011084521 and US Pub No. US20100069426, US20120004293 andUS20100104655, each of which is herein incorporated by reference intheir entirety.

In one embodiment, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. In one embodiment, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968, hereinincorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise amultiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910;each of which is herein incorporated by reference in its entirety).

In one embodiment, the block copolymers described herein may be includedin a polyion complex comprising a non-polymeric micelle and the blockcopolymer. (See e.g., U.S. Pub. No. 20120076836; herein incorporated byreference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference inits entirety) and combinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand. The targeting ligand maybe any ligand known in the art such as, but not limited to, a monoclonalantibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; hereinincorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may be formulated in anaqueous solution which may be used to target cancer (see InternationalPub No. WO2011084513 and US Pub No. US20110294717, each of which isherein incorporated by reference in their entirety).

In one embodiment, the modified nucleic acid molecules or mmRNA may beencapsulated in, linked to and/or associated with syntheticnanocarriers. Synthetic nanocarriers include, but are not limited to,those described in International Pub. Nos. WO2010005740, WO2010030763,WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265,WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405,WO2012149411 and WO2012149454 and US Pub. Nos. US20110262491,US20100104645, US20100087337 and US20120244222, each of which is hereinincorporated by reference in their entirety. The synthetic nanocarriersmay be formulated using methods known in the art and/or describedherein. As a non-limiting example, the synthetic nanocarriers may beformulated by the methods described in International Pub Nos.WO2010005740, WO2010030763 and WO201213501 and US Pub. Nos.US20110262491, US20100104645, US20100087337 and US20120244222, each ofwhich is herein incorporated by reference in their entirety. In anotherembodiment, the synthetic nanocarrier formulations may be lyophilized bymethods described in International Pub. No. WO2011072218 and U.S. Pat.No. 8,211,473; each of which is herein incorporated by reference intheir entirety.

In one embodiment, the synthetic nanocarriers may contain reactivegroups to release the modified nucleic acid molecules and/or mmRNAdescribed herein (see International Pub. No. WO20120952552 and US PubNo. US20120171229, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201, each of which is hereinincorporated by reference in its entirety).

In one embodiment, the synthetic nanocarriers may be formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the modified nucleic acid molecules and/or mmRNAat a specified pH and/or after a desired time interval. As anon-limiting example, the synthetic nanoparticle may be formulated torelease the modified mRNA molecules and/or mmRNA after 24 hours and/orat a pH of 4.5 (see International Pub. Nos. WO2010138193 andWO2010138194 and US Pub Nos. US20110020388 and US20110027217, each ofwhich is herein incorporated by reference in their entirety).

In one embodiment, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the modified nucleic acidmolecules and/or mmRNA described herein. As a non-limiting example, thesynthetic nanocarriers for sustained release may be formulated bymethods known in the art, described herein and/or as described inInternational Pub No. WO2010138192 and US Pub No. 20100303850, each ofwhich is herein incorporated by reference in their entirety.

In one embodiment, the synthetic nanocarrier may be formulated for useas a vaccine. In one embodiment, the synthetic nanocarrier mayencapsulate at least one modified nucleic acid molecule and/or mmRNAwhich encodes at least one antigen. As a non-limiting example, thesynthetic nanocarrier may include at least one antigen and an excipientfor a vaccine dosage form (see International Pub No. WO2011150264 and USPub No. US20110293723, each of which is herein incorporated by referencein their entirety). As another non-limiting example, a vaccine dosageform may include at least two synthetic nanocarriers with the same ordifferent antigens and an excipient (see International Pub No.WO2011150249 and US Pub No. US20110293701, each of which is hereinincorporated by reference in their entirety). The vaccine dosage formmay be selected by methods described herein, known in the art and/ordescribed in International Pub No. WO2011150258 and US Pub No.US20120027806, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the synthetic nanocarrier may comprise at least onemodified nucleic acid molecule and/or mmRNA which encodes at least oneadjuvant. In another embodiment, the synthetic nanocarrier may compriseat least one modified nucleic molecule acid and/or mmRNA and anadjuvant. As a non-limiting example, the synthetic nanocarriercomprising and adjuvant may be formulated by the methods described inInternational Pub No. WO2011150240 and US Pub No. US20110293700, each ofwhich is herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier may encapsulate at leastone modified nucleic acid molecule and/or mmRNA which encodes a peptide,fragment or region from a virus. As a non-limiting example, thesynthetic nanocarrier may include, but is not limited to, thenanocarriers described in International Pub No. WO2012024621,WO201202629, WO2012024632 and US Pub No. US20120064110, US20120058153and US20120058154, each of which is herein incorporated by reference intheir entirety.

In one embodiment, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Pub. No. 20120282343; hereinincorporated by reference in its entirety.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The modified nucleic acid molecules and mmRNA of the invention can beformulated using natural and/or synthetic polymers. Non-limitingexamples of polymers which may be used for delivery include, but are notlimited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena,Calif.) formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison(Madison, Wis.), PHASERX™ polymer formulations such as, withoutlimitation, SMARTT POLYMER TECHNOLOGY™ (Seattle, Wash.), DMRI/DOPE,poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan,cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimersand poly(lactic-co-glycolic acid) (PLGA) polymers, RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as, but not limited to, PHASERX™ (Seattle, Wash.).

A non-limiting example of chitosan formulation includes a core ofpositively charged chitosan and an outer portion of negatively chargedsubstrate (U.S. Pub. No. 20120258176; herein incorporated by referencein its entirety). Chitosan includes, but is not limited to N-trimethylchitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosanderivatives, or combinations thereof.

In one embodiment, the polymers used in the present invention haveundergone processing to reduce and/or inhibit the attachment of unwantedsubstances such as, but not limited to, bacteria, to the surface of thepolymer. The polymer may be processed by methods known and/or describedin the art and/or described in International Pub. No. WO2012150467,herein incorporated by reference in its entirety.

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; herein incorporated by reference in its entirety). Thisparticular approach is a multicomponent polymer system whose keyfeatures include a membrane-active polymer to which nucleic acid, inthis case siRNA, is covalently coupled via a disulfide bond and whereboth PEG (for charge masking) and N-acetylgalactosamine (for hepatocytetargeting) groups are linked via pH-sensitive bonds (Rozema et al., ProcNatl Acad Sci USA. 2007 104:12982-12887; herein incorporated byreference in its entirety). On binding to the hepatocyte and entry intothe endosome, the polymer complex disassembles in the low-pHenvironment, with the polymer exposing its positive charge, leading toendosomal escape and cytoplasmic release of the siRNA from the polymer.Through replacement of the N-acetylgalactosamine group with a mannosegroup, it was shown one could alter targeting from asialoglycoproteinreceptor-expressing hepatocytes to sinusoidal endothelium and Kupffercells. Another polymer approach involves using transferrin-targetedcyclodextrin-containing polycation nanoparticles. These nanoparticleshave demonstrated targeted silencing of the EWS-FLI1 gene product intransferrin receptor-expressing Ewing's sarcoma tumor cells(Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; hereinincorporated by reference in its entirety) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated byreference in its entirety). Both of these delivery strategiesincorporate rational approaches using both targeted delivery andendosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofmodified nucleic acid molecules or mmRNA (e.g., following intramuscularor subcutaneous injection). The altered release profile for the modifiednucleic acid molecule or mmRNA can result in, for example, translationof an encoded protein over an extended period of time. The polymerformulation may also be used to increase the stability of the modifiednucleic acid molecule or mmRNA. Biodegradable polymers have beenpreviously used to protect nucleic acids other than mmRNA fromdegradation and been shown to result in sustained release of payloads invivo (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887;Sullivan et al., Expert Opin Drug Deliv. 2010 7:1433-1446; Convertine etal., Biomacromolecules. 2010 Oct. 1; Chu et al., Acc Chem. Res. 2012Jan. 13; Manganiello et al., Biomaterials. 2012 33:2301-2309; Benoit etal., Biomacromolecules. 2011 12:2708-2714; Singha et al., Nucleic AcidTher. 2011 2:133-147; deFougerolles Hum Gene Ther. 2008 19:125-132;Schaffert and Wagner, Gene Ther. 2008 16:1131-1138; Chaturvedi et al.,Expert Opin Drug Deliv. 2011 8:1455-1468; Davis, Mol. Pharm. 20096:659-668; Davis, Nature 2010 464:1067-1070; each of which is hereinincorporated by reference in its entirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic interaction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol. Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The modified nucleic acid molecules and mmRNA of the invention may beformulated with or in a polymeric compound. The polymer may include atleast one polymer such as, but not limited to, polyethenes, polyethyleneglycol (PEG), poly(1-lysine) (PLL), PEG grafted to PLL, cationiclipopolymer, biodegradable cationic lipopolymer, polyethyleneimine(PEI), cross-linked branched poly(alkylene imines), a polyaminederivative, a modified poloxamer, a biodegradable polymer, elasticbiodegradable polymer, biodegradable block copolymer, biodegradablerandom copolymer, biodegradable polyester copolymer, biodegradablepolyester block copolymer, biodegradable polyester block randomcopolymer, multiblock copolymers, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers, dextran polymers, dextran polymer derivatives or combinationsthereof.

As a non-limiting example, the modified nucleic acid molecules or mmRNAof the invention may be formulated with the polymeric compound of PEGgrafted with PLL as described in U.S. Pat. No. 6,177,274; hereinincorporated by reference in its entirety. The formulation may be usedfor transfecting cells in vitro or for in vivo delivery of the modifiednucleic acid molecules and mmRNA. In another example, the modifiednucleic acid molecules and mmRNA may be suspended in a solution ormedium with a cationic polymer, in a dry pharmaceutical composition orin a solution that is capable of being dried as described in U.S. Pub.Nos. 20090042829 and 20090042825; each of which are herein incorporatedby reference in their entireties.

As another non-limiting example the modified nucleic acid molecules ormmRNA of the invention may be formulated with a PLGA-PEG block copolymer(see US Pub. No. US20120004293 and U.S. Pat. No. 8,236,330, each ofwhich are herein incorporated by reference in their entireties) orPLGA-PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, hereinincorporated by reference in its entirety). As a non-limiting example,the modified nucleic acid molecules or mmRNA of the invention may beformulated with a diblock copolymer of PEG and PLA or PEG and PLGA (seeU.S. Pat. No. 8,246,968, herein incorporated by reference in itsentirety).

A polyamine derivative may be used to deliver nucleic acid moleculesand/or mmRNA or to treat and/or prevent a disease or to be included inan implantable or injectable device (U.S. Pub. No. 20100260817 hereinincorporated by reference in its entirety). As a non-limiting example, apharmaceutical composition may include the modified nucleic acidmolecules and mmRNA and the polyamine derivative described in U.S. Pub.No. 20100260817 (the contents of which are incorporated herein byreference in its entirety). As a non-limiting example the modifiednucleic acids or mmRNA of the present invention may be delivered using apolyaminde polymer such as, but not limited to, a polymer comprising a1,3-dipolar addition polymer prepared by combining a carbohydratediazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat.No. 8,236,280; herein incorporated by reference in its entirety).

The modified nucleic acid molecules and/or mmRNA of the invention may beformulated with at least one acrylic polymer. Acrylic polymers includebut are not limited to, acrylic acid, methacrylic acid, acrylic acid andmethacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethylmethacrylates, cyanoethyl methacrylate, amino alkyl methacrylatecopolymer, poly(acrylic acid), poly(methacrylic acid),polycyanoacrylates and combinations thereof.

In one embodiment, the modified nucleic acid molecules and/or mmRNA ofthe present invention may be formulated with at least one polymer and/orderivatives thereof described in International Publication Nos.WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No.20120283427, each of which are herein incorporated by reference in theirentireties. In another embodiment, the modified nucleic acid moleculesor mmRNA of the present invention may be formulated with a polymer offormula Z as described in WO2011115862, herein incorporated by referencein its entirety. In yet another embodiment, the modified nucleic acidmolecules or mmRNA may be formulated with a polymer of formula Z, Z′ orZ″ as described in International Pub. Nos. WO2012082574 or WO2012068187,each of which are herein incorporated by reference in their entireties.The polymers formulated with the modified nucleic acids and/or modifiedmRNA of the present invention may be synthesized by the methodsdescribed in International Pub. Nos. WO2012082574 or WO2012068187, eachof which are herein incorporated by reference in their entireties.

Formulations of modified nucleic acid molecules and/or mmRNA of theinvention may include at least one amine-containing polymer such as, butnot limited to polylysine, polyethylene imine, poly(amidoamine)dendrimers or combinations thereof.

For example, the modified nucleic acid molecules and/or mmRNA of theinvention may be formulated in a pharmaceutical compound including apoly(alkylene imine), a biodegradable cationic lipopolymer, abiodegradable block copolymer, a biodegradable polymer, or abiodegradable random copolymer, a biodegradable polyester blockcopolymer, a biodegradable polyester polymer, a biodegradable polyesterrandom copolymer, a linear biodegradable copolymer, PAGA, abiodegradable cross-linked cationic multi-block copolymer orcombinations thereof. The biodegradable cationic lipopolymer may be madeby methods known in the art and/or described in U.S. Pat. No. 6,696,038,U.S. App. Nos. 20030073619 and 20040142474 each of which is hereinincorporated by reference in their entireties. The poly(alkylene imine)may be made using methods known in the art and/or as described in U.S.Pub. No. 20100004315, herein incorporated by reference in its entirety.The biodegradabale polymer, biodegradable block copolymer, thebiodegradable random copolymer, biodegradable polyester block copolymer,biodegradable polyester polymer, or biodegradable polyester randomcopolymer may be made using methods known in the art and/or as describedin U.S. Pat. Nos. 6,517,869 and 6,267,987, the contents of which areeach incorporated herein by reference in their entirety. The linearbiodegradable copolymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,652,886. The PAGA polymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,217,912 herein incorporated by reference in its entirety. The PAGApolymer may be copolymerized to form a copolymer or block copolymer withpolymers such as but not limited to, poly-L-lysine, polyargine,polyornithine, histones, avidin, protamines, polylactides andpoly(lactide-co-glycolides). The biodegradable cross-linked cationicmulti-block copolymers may be made my methods known in the art and/or asdescribed in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each ofwhich are herein incorporated by reference in their entireties. Forexample, the multi-block copolymers may be synthesized using linearpolyethyleneimine (LPEI) blocks which have distinct patterns as comparedto branched polyethyleneimines. Further, the composition orpharmaceutical composition may be made by the methods known in the art,described herein, or as described in U.S. Pub. No. 20100004315 or U.S.Pat. Nos. 6,267,987 and 6,217,912 each of which are herein incorporatedby reference in their entireties.

The modified nucleic acid molecules and mmRNA of the invention may beformulated with at least one degradable polyester which may containpolycationic side chains. Degradeable polyesters include, but are notlimited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

The modified nucleic acid molecules and mmRNA of the invention may beformulated with at least one crosslinkable polyester. Crosslinkablepolyesters include those known in the art and described in US Pub. No.20120269761, herein incorporated by reference in its entirety.

In one embodiment, the polymers described herein may be conjugated to alipid-terminating PEG. As a non-limiting example, PLGA may be conjugatedto a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present inventionare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety. The polymers may beconjugated using a ligand conjugate such as, but not limited to, theconjugates described in U.S. Pat. No. 8,273,363, herein incorporated byreference in its entirety.

In one embodiment, the modified nucleic acid molecules and/or mmRNAdescribed herein may be conjugated with another compound. Non-limitingexamples of conjugates are described in U.S. Pat. Nos. 7,964,578 and7,833,992, each of which are herein incorporated by reference in theirentireties. In another embodiment, modified RNA of the present inventionmay be conjugated with conjugates of formula I-122 as described in U.S.Pat. Nos. 7,964,578 and 7,833,992, each of which are herein incorporatedby reference in their entireties. The modified RNA described herein maybe conjugated with a metal such as, but not limited to, gold. (See e.g.,Giljohann et al. Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073; hereinincorporated by reference in its entirety). In another embodiment, themodified nucleic acid molecules and/or mmRNA described herein may beconjugated and/or encapsulated in gold-nanoparticles. (InternationalPub. No. WO201216269 and U.S. Pub. No. 20120302940; each of which isherein incorporated by reference in its entirety).

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the modified nucleicacid and mmRNA of the present invention may be used in a gene deliverycomposition with the poloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), 3B-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HCl) diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The modified nucleic acid molecules and/or mmRNA of the invention may beformulated in a polyplex of one or more polymers (U.S. Pub. No.20120237565 and 20120270927; each of which is herein incorporated byreference in its entirety). In one embodiment, the polyplex comprisestwo or more cationic polymers. The catioinic polymer may comprise apoly(ethylene imine) (PEI) such as linear PEI.

The modified nucleic acid molecules and mmRNA of the invention can alsobe formulated as a nanoparticle using a combination of polymers, lipids,and/or other biodegradable agents, such as, but not limited to, calciumphosphate. Components may be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the modified nucleic acid molecule andmmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller etal., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug DelivRev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Suet al., Mol. Pharm. 2011 Jun. 6; 8(3):774-87; each of which is hereinincorporated by reference in its entirety). As a non-limiting example,the nanoparticle may comprise a plurality of polymers such as, but notlimited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA),hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers(International Pub. No. WO20120225129; herein incorporated by referencein its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver modified nucleic acidmolecules and mmRNA in vivo. In one embodiment, a lipid coated calciumphosphate nanoparticle, which may also contain a targeting ligand suchas anisamide, may be used to deliver the modified nucleic acid moleculeand mmRNA of the present invention. For example, to effectively deliversiRNA in a mouse metastatic lung model a lipid coated calcium phosphatenanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421; Li etal., J Contr Rel. 2012 158:108-114; Yang et al., Mol. Ther. 201220:609-615; herein incorporated by reference in its entirety). Thisdelivery system combines both a targeted nanoparticle and a component toenhance the endosomal escape, calcium phosphate, in order to improvedelivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to deliver modified nucleic acid molecules andmmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., JContr Rel. 2006 111:368-370; herein incorporated by reference in itsentirety).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the modified nucleic acid molecules and mmRNA of the presentinvention. The PEG-charge-conversional polymer may improve upon thePEG-polyanion block copolymers by being cleaved into a polycation atacidic pH, thus enhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG may be used to deliveryof the modified nucleic acid molecules and mmRNA of the presentinvention. As a non-limiting example, in mice bearing aluciferease-expressing tumor, it was determined that thelipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031; herein incorporated byreference in its entirety).

In one embodiment, the lipid nanoparticles may comprise a core of themodified nucleic acid molecules disclosed herein and a polymer shell.The polymer shell may be any of the polymers described herein and areknown in the art. In an additional embodiment, the polymer shell may beused to protect the modified nucleic acids in the core.

Core-shell nanoparticles for use with the modified nucleic acidmolecules of the present invention are described and may be formed bythe methods described in U.S. Pat. No. 8,313,777 herein incorporated byreference in its entirety.

In one embodiment, the core-shell nanoparticles may comprise a core ofthe modified nucleic acid molecules disclosed herein and a polymershell. The polymer shell may be any of the polymers described herein andare known in the art. In an additional embodiment, the polymer shell maybe used to protect the modified nucleic acid molecules in the core.

Peptides and Proteins

The modified nucleic acid molecules and mmRNA of the invention can beformulated with peptides and/or proteins in order to increasetransfection of cells by the modified nucleic acid molecules or mmRNA.In one embodiment, peptides such as, but not limited to, cellpenetrating peptides and proteins and peptides that enable intracellulardelivery may be used to deliver pharmaceutical formulations. Anon-limiting example of a cell penetrating peptide which may be usedwith the pharmaceutical formulations of the present invention include acell-penetrating peptide sequence attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel,Cell-Penetrating Peptides Processes and Applications (CRC Press, BocaRaton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life. Sci.62(16):1839-49 (2005), all of which are incorporated herein byreference). The compositions can also be formulated to include a cellpenetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. Modified nucleic acid moleculesand mmRNA of the invention may be complexed to peptides and/or proteinssuch as, but not limited to, peptides and/or proteins from AileronTherapeutics (Cambridge, Mass.) and Permeon Biologics (Cambridge, Mass.)in order to enable intracellular delivery (Cronican et al., ACS Chem.Biol. 2010 5:747-752; McNaughton et al., Proc. Natl. Acad. Sci. USA 2009106:6111-6116; Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine andHilinski, Methods Enzymol. 2012; 503:3-33; all of which are hereinincorporated by reference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the modified nucleic acid molecules or mmRNA may beintroduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the modified nucleic acid molecule ormmRNA, alter the biodistribution of the modified nucleic acid moleculeor mmRNA (e.g., by targeting specific tissues or cell types), and/orincrease the translation of encoded protein. (See e.g., InternationalPub. No. WO2012110636; herein incorporated by reference in itsentirety).

Cells

The modified nucleic acid molecule and mmRNA of the invention can betransfected ex vivo into cells, which are subsequently transplanted intoa subject. As non-limiting examples, the pharmaceutical compositions mayinclude red blood cells to deliver modified RNA to liver and myeloidcells, virosomes to deliver modified nucleic acid molecules and mmRNA invirus-like particles (VLPs), and electroporated cells such as, but notlimited to, from MAXCYTE® (Gaithersburg, Md.) and from ERYTECH® (Lyon,France) to deliver modified RNA. Examples of use of red blood cells,viral particles and electroporated cells to deliver payloads other thanmmRNA have been documented (Godfrin et al., Expert Opin Biol Ther. 201212:127-133; Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu etal., Proc Natl Acad Sci USA. 2011 108:10980-10985; Lund et al., PharmRes. 2010 27:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47;Cusi, Hum Vaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 200613:400-411; all of which are herein incorporated by reference in itsentirety). The modified nucleic acid molecules and mmRNA may bedelivered in synthetic VLPs synthesized by the methods described inInternational Pub No. WO2011085231 and US Pub No. 20110171248, each ofwhich are herein incorporated by reference in their entireties.

Cell-based formulations of the modified nucleic acid molecules and mmRNAof the invention may be used to ensure cell transfection (e.g., in thecellular carrier), alter the biodistribution of the modified nucleicacid molecule or mmRNA (e.g., by targeting the cell carrier to specifictissues or cell types), and/or increase the translation of encodedprotein.

Introduction into Cells

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporaiton, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are taught for example as it relates to bacteria in US PatentPublication 20100196983 and as it relates to other cell types in, forexample, US Patent Publication 20100009424, each of which areincorporated herein by reference in their entirety.

Electroporation techniques are also well known in the art. In oneembodiment, modified nucleic acid molecules or mmRNA may be delivered byelectroporation as described in Example 8.

Hyaluronidase

The intramuscular or subcutaneous localized injection of modifiednucleic acid molecules or mmRNA of the invention can includehyaluronidase, which catalyzes the hydrolysis of hyaluronan. Bycatalyzing the hydrolysis of hyaluronan, a constituent of theinterstitial barrier, hyaluronidase lowers the viscosity of hyaluronan,thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv.(2007) 4:427-440; herein incorporated by reference in its entirety). Itis useful to speed their dispersion and systemic distribution of encodedproteins produced by transfected cells. Alternatively, the hyaluronidasecan be used to increase the number of cells exposed to a modifiednucleic acid molecule or mmRNA of the invention administeredintramuscularly or subcutaneously.

Nanoparticle Mimics

The modified nucleic acid molecules and mmRNA of the invention may beencapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example themodified mRNA of the invention may be encapsulated in a non-vironparticle which can mimic the delivery function of a virus (seeInternational Pub. No. WO2012006376 herein incorporated by reference inits entirety).

Nanotubes

The modified nucleic acid molecules or mmRNA of the invention can beattached or otherwise bound to at least one nanotube such as, but notlimited to, rosette nanotubes, rosette nanotubes having twin bases witha linker, carbon nanotubes and/or single-walled carbon nanotubes, Themodified nucleic acid molecules or mmRNA may be bound to the nanotubesthrough forces such as, but not limited to, steric, ionic, covalentand/or other forces.

In one embodiment, the nanotube can release one or more modified nucleicacid molecule or mmRNA into cells. The size and/or the surface structureof at least one nanotube may be altered so as to govern the interactionof the nanotubes within the body and/or to attach or bind to themodified nucleic acid molecule or mmRNA disclosed herein. In oneembodiment, the building block and/or the functional groups attached tothe building block of the at least one nanotube may be altered to adjustthe dimensions and/or properties of the nanotube. As a non-limitingexample, the length of the nanotubes may be altered to hinder thenanotubes from passing through the holes in the walls of normal bloodvessels but still small enough to pass through the larger holes in theblood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the modified mRNA may be mixed with pharmaceuticallyacceptable excipients and/or delivery vehicles.

In one embodiment, the modified mRNA are attached and/or otherwise boundto at least one rosette nanotube. The rosette nanotubes may be formed bya process known in the art and/or by the process described inInternational Publication No. WO2012094304, herein incorporated byreference in its entirety. At least one modified mRNA may be attachedand/or otherwise bound to at least one rosette nanotube by a process asdescribed in International Publication No. WO2012094304, hereinincorporated by reference in its entirety, where rosette nanotubes ormodules forming rosette nanotubes are mixed in aqueous media with atleast one modified mRNA under conditions which may cause at least onemodified mRNA to attach or otherwise bind to the rosette nanotubes.

In one embodiment, the modified nucleic acid molecule or mmRNA may beattached to and/or otherwise bound to at least one carbon nanotube. As anon-limiting example, the modified nucleic acid molecule or mmRNA may bebound to a linking agent and the linked agent may be bound to the carbonnanotube (See e.g., U.S. Pat. No. 8,246,995; herein incorporated byreference in its entirety). The carbon nanotube may be a single-wallednanotube (See e.g., U.S. Pat. No. 8,246,995; herein incorporated byreference in its entirety).

Conjugates

The modified nucleic acids molecules and mmRNA of the invention includeconjugates, such as a modified nucleic acid molecule or mmRNA covalentlylinked to a carrier or targeting group, or including two encodingregions that together produce a fusion protein (e.g., bearing atargeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the modified nucleic acid molecules and mmRNA of thepresent invention. The conjugate may comprise a cationic polymer suchas, but not limited to, polyamine, polylysine, polyalkylenimine, andpolyethylenimine which may be grafted to with poly(ethylene glycol). Asa non-limiting example, the conjugate may be similar to the polymericconjugate and the method of synthesizing the polymeric conjugatedescribed in U.S. Pat. No. 6,586,524 herein incorporated by reference inits entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include modified nucleicacids or mmRNA with phosphorothioate backbones and oligonucleosides withother modified backbones, and in particular —CH₂—NH—CH₂—,—CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—and —N(CH₃)—CH₂—CH₂-[whereinthe native phosphodiester backbone is represented as —O—P(O)₂—O—CH₂—] ofthe above-referenced U.S. Pat. No. 5,489,677, and the amide backbones ofthe above-referenced U.S. Pat. No. 5,602,240. In some embodiments, thepolynucletotides featured herein have morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′ UTR, a 3′ UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(b)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(m)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)O[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the modified nucleicacids or mmRNA include one of the following at the 2′ position: C₁ toC₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties, or a group for improving the pharmacodynamic properties, andother substituents having similar properties. In some embodiments, themodification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also knownas 2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. Polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which isherein incorporated by reference.

In still other embodiments, the modified nucleic acid molecule or mmRNAis covalently conjugated to a cell-penetrating polypeptide. Thecell-penetrating peptide may also include a signal sequence. Theconjugates of the invention can be designed to have increased stability;increased cell transfection; and/or altered the biodistribution (e.g.,targeted to specific tissues or cell types).

Self-Assembled Nanoparticles Nucleic Acid Self-Assembled Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles were prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393; herein incorporated by referencein its entirety).

In one embodiment, the modified nucleic acid molecules and mmRNAdisclosed herein may be formulated as self-assembled nanoparticles. As anon-limiting example, nucleic acids may be used to make nanoparticleswhich may be used in a delivery system for the modified nucleic acidmolecules and/or mmRNA of the present invention (See e.g., InternationalPub. No. WO2012125987; herein incorporated by reference in itsentirety).

In one embodiment, the nucleic acid self-assembled nanoparticles maycomprise a core of the modified nucleic acid molecules or mmRNAdisclosed herein and a polymer shell. The polymer shell may be any ofthe polymers described herein and are known in the art. In an additionalembodiment, the polymer shell may be used to protect the modifiednucleic acid molecules and mmRNA in the core.

Polymer-Based Self-Assembled Nanoparticles

Polymers may be used to form sheets which self-assembled intonanoparticles. These nanoparticles may be used to deliver the modifiednucleic acids and mmRNA of the present invention. In one embodiment,these self-assembled nanoparticles may be microsponges formed of longpolymers of RNA hairpins which form into crystalline ‘pleated’ sheetsbefore self-assembling into microsponges. These microsponges aredensely-packed sponge like microparticles which may function as anefficient carrier and may be able to deliver cargo to a cell. Themicrosponges may be from 1 um to 300 nm in diameter. The microspongesmay be complexed with other agents known in the art to form largermicrosponges. As a non-limiting example, the microsponge may becomplexed with an agent to form an outer layer to promote cellularuptake such as polycation polyethyleneime (PEI). This complex can form a250-nm diameter particle that can remain stable at high temperatures(150° C.) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; hereinincorporated by reference in its entirety). Additionally thesemicrosponges may be able to exhibit an extraordinary degree ofprotection from degradation by ribonucleases.

In another embodiment, the polymer-based self-assembled nanoparticlessuch as, but not limited to, microsponges, may be fully programmablenanoparticles. The geometry, size and stoichiometry of the nanoparticlemay be precisely controlled to create the optimal nanoparticle fordelivery of cargo such as, but not limited to, modified nucleic acidmolecules and mmRNA.

In one embodiment, the polymer based nanoparticles may comprise a coreof the modified nucleic acid molecules and mmRNA disclosed herein and apolymer shell. The polymer shell may be any of the polymers describedherein and are known in the art. In an additional embodiment, thepolymer shell may be used to protect the modified nucleic acid moleculesand mmRNA in the core.

Inorganic Nanoparticles

The modified nucleic acid molecules or mmRNAs of the present inventionmay be formulated in inorganic nanoparticles (U.S. Pat. No. 8,257,745,herein incorporated by reference in its entirety). The inorganicnanoparticles may include, but are not limited to, clay substances thatare water swellable. As a non-limiting example, the inorganicnanoparticle may include synthetic smectite clays which are made fromsimple silicates (See e.g., U.S. Pat. Nos. 5,585,108 and 8,257,745 eachof which are herein incorporated by reference in their entirety).

In one embodiment, the inorganic nanoparticles may comprise a core ofthe modified nucleic acids disclosed herein and a polymer shell. Thepolymer shell may be any of the polymers described herein and are knownin the art. In an additional embodiment, the polymer shell may be usedto protect the modified nucleic acids in the core.

Semi-Conductive and Metallic Nanoparticles

The modified nucleic acid molecules or mmRNAs of the present inventionmay be formulated in water-dispersible nanoparticle comprising asemiconductive or metallic material (U.S. Pub. No. 20120228565; hereinincorporated by reference in its entirety) or formed in a magneticnanoparticle (U.S. Pub. No. 20120265001 and 20120283503; each of whichis herein incorporated by reference in its entirety). Thewater-dispersible nanoparticles may be hydrophobic nanoparticles orhydrophilic nanoparticles.

In one embodiment, the semi-conductive and/or metallic nanoparticles maycomprise a core of the modified nucleic acids disclosed herein and apolymer shell. The polymer shell may be any of the polymers describedherein and are known in the art. In an additional embodiment, thepolymer shell may be used to protect the modified nucleic acids in thecore.

Gels and Hydrogels

In one embodiment, the modified mRNA disclosed herein may beencapsulated into any hydrogel known in the art which may form a gelwhen injected into a subject. Hydrogels are a network of polymer chainsthat are hydrophilic, and are sometimes found as a colloidal gel inwhich water is the dispersion medium. Hydrogels are highly absorbent(they can contain over 99% water) natural or synthetic polymers.Hydrogels also possess a degree of flexibility very similar to naturaltissue, due to their significant water content. The hydrogel describedherein may used to encapsulate lipid nanoparticles which arebiocompatible, biodegradable and/or porous.

As a non-limiting example, the hydrogel may be an aptamer-functionalizedhydrogel. The aptamer-functionalized hydrogel may be programmed torelease one or more modified nucleic acid molecules and/or mmRNA usingnucleic acid hybridization. (Battig et al., J. Am. Chem. Society. 2012134:12410-12413; herein incorporated by reference in its entirety).

As another non-limiting example, the hydrogel may be a shaped as aninverted opal. The opal hydrogels exhibit higher swelling ratios and theswelling kinetics is an order of magnitude faster as well. Methods ofproducing opal hydrogels and description of opal hydrogels are describedin International Pub. No. WO2012148684, herein incorporated by referencein its entirety.

In yet another non-limiting example, the hydrogel may be anantibacterial hydrogel. The antibacterial hydrogel may comprise apharmaceutical acceptable salt or organic material such as, but notlimited to pharmaceutical grade and/or medical grade silver salt andaloe vera gel or extract. (International Pub. No. WO2012151438, hereinincorporated by reference in its entirety).

In one embodiment, the modified mRNA may be encapsulated in a lipidnanoparticle and then the lipid nanoparticle may be encapsulated into ahydrogel.

In one embodiment, the modified mRNA disclosed herein may beencapsulated into any gel known in the art. As a non-limiting examplethe gel may be a fluorouracil injectable gel or a fluorouracilinjectable gel containing a chemical compound and/or drug known in theart. As another example, the modified mRNA may be encapsulated in afluorouracil gel containing epinephrine (See e.g., Smith et al. CancerChemotherapy and Pharmacology, 1999 44(4):267-274; herein incorporatedby reference in its entirety).

In one embodiment, the modified nucleic acid molecules and/or mmRNAdisclosed herein may be encapsulated into a fibrin gel, fibrin hydrogelor fibrin glue. In another embodiment, the modified nucleic acidmolecules and/or mmRNA may be formulated in a lipid nanoparticle or arapidly eliminated lipid nanoparticle prior to being encapsulated into afibrin gel, fibrin hydrogel or a fibrin glue. In yet another embodiment,the modified nucleic acid molecules and/or mmRNA may be formulated as alipoplex prior to being encapsulated into a fibrin gel, hydrogel or afibrin glue. Fibrin gels, hydrogels and glues comprise two components, afibrinogen solution and a thrombin solution which is rich in calcium(See e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148:49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; eachof which is herein incorporated by reference in its entirety). Theconcentration of the components of the fibrin gel, hydrogel and/or gluecan be altered to change the characteristics, the network mesh size,and/or the degradation characteristics of the gel, hydrogel and/or gluesuch as, but not limited to changing the release characteristics of thefibrin gel, hydrogel and/or glue. (See e.g., Spicer and Mikos, Journalof Controlled Release 2010. 148: 49-55; Kidd et al. Journal ofControlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering2008. 14:119-128; each of which is herein incorporated by reference inits entirety). This feature may be advantageous when used to deliver themodified mRNA disclosed herein. (See e.g., Kidd et al. Journal ofControlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering2008. 14:119-128; each of which is herein incorporated by reference inits entirety).

Cations and Anions

Formulations of modified nucleic acid molecules disclosed herein mayinclude cations or anions. In one embodiment, the formulations includemetal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ andcombinations thereof. As a non-limiting example, formulations mayinclude polymers and a modified mRNA complexed with a metal cation (Seee.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which is hereinincorporated by reference in its entirety).

Molded Nanoparticles and Microparticles

The modified nucleic acid molecules and/or mmRNA disclosed herein may beformulated in nanoparticles and/or microparticles. These nanoparticlesand/or microparticles may be molded into any size shape and chemistry.As an example, the nanoparticles and/or microparticles may be made usingthe PRINT° technology by LIQUIDA TECHNOLOGIES® (Morrisville, N.C.) (Seee.g., International Pub. No. WO2007024323; herein incorporated byreference in its entirety).

In one embodiment, the molded nanoparticles may comprise a core of themodified nucleic acid molecules and/or mmRNA disclosed herein and apolymer shell. The polymer shell may be any of the polymers describedherein and are known in the art. In an additional embodiment, thepolymer shell may be used to protect the modified nucleic acid moleculesand/or mmRNA in the core.

NanoJackets and NanoLiposomes

The modified nucleic acid molecules and/or mmRNA disclosed herein may beformulated in NanoJackets and NanoLiposomes by Keystone Nano (StateCollege, Pa.). NanoJackets are made of compounds that are naturallyfound in the body including calcium, phosphate and may also include asmall amount of silicates. Nanojackets may range in size from 5 to 50 nmand may be used to deliver hydrophilic and hydrophobic compounds suchas, but not limited to, modified nucleic acid molecules and/or mmRNA.

NanoLiposomes are made of lipids such as, but not limited to, lipidswhich naturally occur in the body. NanoLiposomes may range in size from60-80 nm and may be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, modified nucleic acid moleculesand/or mmRNA. In one aspect, the modified nucleic acids disclosed hereinare formulated in a NanoLiposome such as, but not limited to, CeramideNanoLiposomes.

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but are notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Various excipients for formulating pharmaceuticalcompositions and techniques for preparing the composition are known inthe art (see Remington: The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference in its entirety). The use of aconventional excipient medium may be contemplated within the scope ofthe present disclosure, except insofar as any conventional excipientmedium may be incompatible with a substance or its derivatives, such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition.

In some embodiments, a pharmaceutically acceptable excipient may be atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, an excipient may be approved for use forhumans and for veterinary use. In some embodiments, an excipient may beapproved by United States Food and Drug Administration. In someembodiments, an excipient may be of pharmaceutical grade. In someembodiments, an excipient may meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.The composition may also include excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of modified nucleic acidmolecules or mmRNA for any of therapeutic, pharmaceutical, diagnostic orimaging by any appropriate route taking into consideration likelyadvances in the sciences of drug delivery. Delivery may be naked orformulated.

Naked Delivery

The modified nucleic acid molecules or mmRNA of the present inventionmay be delivered to a cell naked. As used herein in, “naked” refers todelivering modified nucleic acid molecules or mmRNA free from agentswhich promote transfection. For example, the modified nucleic acidmolecules or mmRNA delivered to the cell may contain no modifications.The naked modified nucleic acid molecules or mmRNA may be delivered tothe cell using routes of administration known in the art and describedherein.

Formulated Delivery

The modified nucleic acid molecules or mmRNA of the present inventionmay be formulated, using the methods described herein. The formulationsmay contain modified nucleic acid molecules or mmRNA which may bemodified and/or unmodified. The formulations may further include, butare not limited to, cell penetration agents, a pharmaceuticallyacceptable carrier, a delivery agent, a bioerodible or biocompatiblepolymer, a solvent, and a sustained-release delivery depot. Theformulated modified nucleic acid molecules or mmRNA may be delivered tothe cell using routes of administration known in the art and describedherein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The modified nucleic acid molecules or mmRNA of the present inventionmay be administered by any route which results in a therapeuticallyeffective outcome. These include, but are not limited to enteral,gastroenteral, epidural, oral, transdermal, epidural (peridural),intracerebral (into the cerebrum), intracerebroventricular (into thecerebral ventricles), epicutaneous (application onto the skin),intradermal, (into the skin itself), subcutaneous (under the skin),nasal administration (through the nose), intravenous (into a vein),intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the modified nucleic acids ormmRNA of the present invention are described below.

Parenteral and Injectible Administration

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, oral compositions can include adjuvants such as wettingagents, emulsifying and suspending agents, sweetening, flavoring, and/orperfuming agents. In certain embodiments for parenteral administration,compositions are mixed with solubilizing agents such as CREMOPHOR®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, and/or elixirs. In addition to active ingredients,liquid dosage forms may comprise inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and/or perfuming agents.In certain embodiments for parenteral administration, compositions aremixed with solubilizing agents such as CREMOPHOR®, alcohols, oils,modified oils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the modified nucleic acidmolecules or mmRNA of the invention may be formulated for administrationtopically. The skin may be an ideal target site for delivery as it isreadily accessible. Gene expression may be restricted not only to theskin, potentially avoiding nonspecific toxicity, but also to specificlayers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver modified nucleic acid molecules or mmRNA to theskin: (i) topical application (e.g. for local/regional treatment); (ii)intradermal injection (e.g. for local/regional treatment); and (iii)systemic delivery (e.g. for treatment of dermatologic diseases thataffect both cutaneous and extracutaneous regions). Modified nucleic acidmolecules or mmRNA can be delivered to the skin by several differentapproaches known in the art. Most topical delivery approaches have beenshown to work for delivery of DNA, such as but not limited to, topicalapplication of non-cationic liposome-DNA complex, cationic liposome-DNAcomplex, particle-mediated (gene gun), puncture-mediated genetransfections, and viral delivery approaches. After delivery of thenucleic acid, gene products have been detected in a number of differentskin cell types, including, but not limited to, basal keratinocytes,sebaceous gland cells, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or modified nucleic acidmolecules or mmRNA described herein to allow a user to perform multipletreatments of a subject(s).

In one embodiment, the invention provides for the modified nucleic acidmolecules or mmRNA compositions to be delivered in more than oneinjection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; each of which are herein incorporatedby reference in their entireties. Methods of enhancing the permeabilityof tissue are described in U.S. Publication Nos. 20040171980 and20040236268 and U.S. Pat. No. 6,190,315; each of which are hereinincorporated by reference in their entireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein.The permeability of skin may be measured by methods known in the artand/or described in U.S. Pat. No. 6,190,315, herein incorporated byreference in its entirety. As a non-limiting example, a modified mRNAformulation may be delivered by the drug delivery methods described inU.S. Pat. No. 6,190,315, herein incorporated by reference in itsentirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein,which may further contain a substance that invokes an immune response.In another non-limiting example, a formulation containing a substance toinvoke an immune response may be delivered by the methods described inU.S. Publication Nos. 20040171980 and 20040236268; each of which areherein incorporated by reference in their entireties.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required.

Additionally, the present invention contemplates the use of transdermalpatches, which often have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms may be prepared,for example, by dissolving and/or dispensing the compound in the propermedium. Alternatively or additionally, rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the compoundin a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the modified nucleic acid molecules ormmRNA are spatially retained within or proximal to a target tissue.Provided are method of providing a composition to a target tissue of amammalian subject by contacting the target tissue (which contains one ormore target cells) with the composition under conditions such that thecomposition, in particular the nucleic acid component(s) of thecomposition, is substantially retained in the target tissue, meaningthat at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98,99, 99.9, 99.99 or greater than 99.99% of the composition is retained inthe target tissue. Advantageously, retention is determined by measuringthe amount of the nucleic acid present in the composition that entersone or more target cells. For example, at least 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than99.99% of the nucleic acids administered to the subject are presentintracellularly at a period of time following administration. Forexample, intramuscular injection to a mammalian subject is performedusing an aqueous composition containing a ribonucleic acid and atransfection reagent, and retention of the composition is determined bymeasuring the amount of the ribonucleic acid present in the musclecells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. The composition contains an effective amount of anucleic acid molecules or mmRNA such that the polypeptide of interest isproduced in at least one target cell. The compositions generally containa cell penetration agent, although “naked” nucleic acid (such as nucleicacids without a cell penetration agent or other agent) is alsocontemplated, and a pharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains modified nucleic acid molecule or mmRNA characterized inthat a unit quantity of composition has been determined to produce thepolypeptide of interest in a substantial percentage of cells containedwithin a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentmodified nucleic acid molecules or mmRNA, where one or more than one ofthe modified nucleic acid molecules or mmRNA encodes a polypeptide ofinterest. Optionally, the composition also contains a cell penetrationagent to assist in the intracellular delivery of the composition. Adetermination is made of the dose of the composition required to producethe polypeptide of interest in a substantial percentage of cellscontained within the predetermined volume of the target tissue(generally, without inducing significant production of the polypeptideof interest in tissue adjacent to the predetermined volume, or distallyto the target tissue). Subsequent to this determination, the determineddose is introduced directly into the tissue of the mammalian subject.

In one embodiment, the invention provides for the modified nucleic acidmolecules or mmRNA to be delivered in more than one injection or bysplit dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir, patch pump or osmotic pump.Non-limiting examples of patch pumps include those manufactured and/orsold by BD®, (Franklin Lakes, N.J.), Insulet Corporation (Bedford,Mass.), SteadyMed Therapeutics (San Francisco, Calif.), Medtronic(Minneapolis, Minn.) (e.g., MiniMed), UniLife (York, Pa.), Valeritas(Bridgewater, N.J.), and SpringLeaf Therapeutics (Boston, Mass.). Anon-limiting example of an osmotic pump include those manufactured byDURECT® (Cupertino, Calif.) (e.g., DUROS® and ALZET®).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

As a non-limiting example, the modified nucleic acid molecules or mmRNAdescribed herein may be formulated for pulmonary delivery by the methodsdescribed in U.S. Pat. No. 8,257,685; herein incorporated by referencein its entirety.

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention. A multilayer thin film device may be prepared to containa pharmaceutical composition for delivery to the eye and/or surroundingtissue.

Payload Administration Detectable Agents and Therapeutic Agents

The modified nucleic acid molecules or mmRNA described herein can beused in a number of different scenarios in which delivery of a substance(the “payload”) to a biological target is desired, for example deliveryof detectable substances for detection of the target, or delivery of atherapeutic agent. Detection methods can include, but are not limitedto, both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The modified nucleic acid molecules or mmRNA can be designed to includeboth a linker and a payload in any useful orientation. For example, alinker having two ends is used to attach one end to the payload and theother end to the nucleobase, such as at the C-7 or C-8 positions of thedeaza-adenosine or deaza-guanosine or to the N-3 or C-5 positions ofcytosine or uracil. The polynucleotide of the invention can include morethan one payload (e.g., a label and a transcription inhibitor), as wellas a cleavable linker.

In one embodiment, the modified nucleotide is a modified7-deaza-adenosine triphosphate, where one end of a cleavable linker isattached to the C7 position of 7-deaza-adenine, the other end of thelinker is attached to an inhibitor (e.g., to the C5 position of thenucleobase on a cytidine), and a label (e.g., Cy5) is attached to thecenter of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless inFIG. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporatedherein by reference). Upon incorporation of the modified7-deaza-adenosine triphosphate to an encoding region, the resultingpolynucleotide having a cleavable linker attached to a label and aninhibitor (e.g., a polymerase inhibitor). Upon cleavage of the linker(e.g., with reductive conditions to reduce a linker having a cleavabledisulfide moiety), the label and inhibitor are released. Additionallinkers and payloads (e.g., therapeutic agents, detectable labels, andcell penetrating payloads) are described herein.

Scheme 12 below depicts an exemplary modified nucleotide wherein thenucleobase, adenine, is attached to a linker at the C-7 carbon of7-deaza adenine. In addition, Scheme 12 depicts the modified nucleotidewith the linker and payload, e.g., a detectable agent, incorporated ontothe 3′ end of the mRNA. Disulfide cleavage and 1,2-addition of the thiolgroup onto the propargyl ester releases the detectable agent. Theremaining structure (depicted, for example, as pApC5 Parg in Scheme 12)is the inhibitor. The rationale for the structure of the modifiednucleotides is that the tethered inhibitor sterically interferes withthe ability of the polymerase to incorporate a second base. Thus, it iscritical that the tether be long enough to affect this function and thatthe inhibiter be in a stereochemical orientation that inhibits orprohibits second and follow on nucleotides into the growingpolynucleotide strand.

For example, the modified nucleic acid molecules or mmRNA describedherein can be used in reprogramming induced pluripotent stem cells (iPScells), which can directly track cells that are transfected compared tototal cells in the cluster. In another example, a drug that may beattached to the modified nucleic acid molecules or mmRNA via a linkerand may be fluorescently labeled can be used to track the drug in vivo,e.g. intracellularly. Other examples include, but are not limited to,the use of modified nucleic acid molecules or mmRNA in reversible drugdelivery into cells.

The modified nucleic acid molecules or mmRNA described herein can beused in intracellular targeting of a payload, e.g., detectable ortherapeutic agent, to specific organelle. Exemplary intracellulartargets can include, but are not limited to, the nuclear localizationfor advanced mRNA processing, or a nuclear localization sequence (NLS)linked to the mRNA containing an inhibitor.

In addition, the modified nucleic acid molecules or mmRNA describedherein can be used to deliver therapeutic agents to cells or tissues,e.g., in living animals. For example, the modified nucleic acids ormmRNA described herein can be used to deliver highly polarchemotherapeutics agents to kill cancer cells. The modified nucleic acidmolecules or mmRNA attached to the therapeutic agent through a linkercan facilitate member permeation allowing the therapeutic agent totravel into a cell to reach an intracellular target.

In one example, the linker is attached at the 2′-position of the ribosering and/or at the 3′ and/or 5′ position of the modified nucleic acidmolecule or mmRNA (See e.g., International Pub. No. WO2012030683, hereinincorporated by reference in its entirety). The linker may be any linkerdisclosed herein, known in the art and/or disclosed in InternationalPub. No. WO2012030683, herein incorporated by reference in its entirety.

In another example, the modified nucleic acid molecules or mmRNA can beattached to the modified nucleic acid molecules or mmRNA a viralinhibitory peptide (VIP) through a cleavable linker. The cleavablelinker can release the VIP and dye into the cell. In another example,the modified nucleic acid molecules or mmRNA can be attached through thelinker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H or ^(99m)Tc (e.g., as pertechnetate (technetate(VII),TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., gold nanoparticles),gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagneticiron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), andultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates(e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexyl),microbubbles, or perfluorocarbons). Such optically-detectable labelsinclude for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and -6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (Cibacron™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectablepre-cursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.Combinations

The nucleic acid molecules or mmRNA may be used in combination with oneor more other therapeutic, prophylactic, diagnostic, or imaging agents.By “in combination with,” it is not intended to imply that the agentsmust be administered at the same time and/or formulated for deliverytogether, although these methods of delivery are within the scope of thepresent disclosure. Compositions can be administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. In general, each agent will be administered at adose and/or on a time schedule determined for that agent. In someembodiments, the present disclosure encompasses the delivery ofpharmaceutical, prophylactic, diagnostic, or imaging compositions incombination with agents that may improve their bioavailability, reduceand/or modify their metabolism, inhibit their excretion, and/or modifytheir distribution within the body. As a non-limiting example, thenucleic acid molecules or mmRNA may be used in combination with apharmaceutical agent for the treatment of cancer or to controlhyperproliferative cells. In U.S. Pat. No. 7,964,571, hereinincorporated by reference in its entirety, a combination therapy for thetreatment of solid primary or metastasized tumor is described using apharmaceutical composition including a DNA plasmid encoding forinterleukin-12 with a lipopolymer and also administering at least oneanticancer agent or chemotherapeutic. Further, the nucleic acidmolecules and mmRNA of the present invention that encodesanti-proliferative molecules may be in a pharmaceutical composition witha lipopolymer (see e.g., U.S. Pub. No. 20110218231, herein incorporatedby reference in its entirety, claiming a pharmaceutical compositioncomprising a DNA plasmid encoding an anti-proliferative molecule and alipopolymer) which may be administered with at least onechemotherapeutic or anticancer agent.

Cell Penetrating Payloads

In some embodiments, the modified nucleotides and modified nucleic acidmolecules, which are incorporated into a nucleic acid, e.g., RNA ormRNA, can also include a payload that can be a cell penetrating moietyor agent that enhances intracellular delivery of the compositions. Forexample, the compositions can include, but are not limited to, acell-penetrating peptide sequence that facilitates delivery to theintracellular space, e.g., HIV-derived TAT peptide, penetratins,transportans, or hCT derived cell-penetrating peptides, see, e.g., Caronet al., (2001) Mol. Ther. 3(3):310-8; Langel, Cell-Penetrating Peptides:Processes and Applications (CRC Press, Boca Raton Fla. 2002);El-Andaloussi et al., (2005) Curr Pharm Des. 11(28):3597-611; andDeshayes et al., (2005) Cell Mol Life Sci. 62(16):1839-49; all of whichare incorporated herein by reference. The compositions can also beformulated to include a cell penetrating agent, e.g., liposomes, whichenhance delivery of the compositions to the intracellular space.

Biological Targets

The modified nucleotides and modified nucleic acid molecules describedherein, which are incorporated into a nucleic acid, e.g., RNA or mRNA,can be used to deliver a payload to any biological target for which aspecific ligand exists or can be generated. The ligand can bind to thebiological target either covalently or non-covalently.

Examples of biological targets include, but are not limited to,biopolymers, e.g., antibodies, nucleic acids such as RNA and DNA,proteins, enzymes; examples of proteins include, but are not limited to,enzymes, receptors, and ion channels. In some embodiments the target maybe a tissue- or a cell-type specific marker, e.g., a protein that isexpressed specifically on a selected tissue or cell type. In someembodiments, the target may be a receptor, such as, but not limited to,plasma membrane receptors and nuclear receptors; more specific examplesinclude, but are not limited to, G-protein-coupled receptors, cell poreproteins, transporter proteins, surface-expressed antibodies, HLAproteins, MHC proteins and growth factor receptors.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

According to the present invention, it has been discovered thatadministration of mmRNA in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administeredin one dose/at one time/single route/single point of contact, i.e.,single administration event. As used herein, a “total daily dose” is anamount given or prescribed in 24 hr period. It may be administered as asingle unit dose. In one embodiment, the mmRNA of the present inventionare administered to a subject in split doses. The mmRNA may beformulated in buffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, water, Ringer'ssolution, U.S.P., and isotonic sodium chloride solution. Sterile, fixedoils are conventionally employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. Fatty acids such as oleic acid can be used in thepreparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of modified mRNA thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered modified mRNA may be accomplished bydissolving or suspending the modified mRNA in an oil vehicle. Injectabledepot forms are made by forming microencapsule matrices of the modifiedmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of modified mRNA to polymer and the nature ofthe particular polymer employed, the rate of modified mRNA release canbe controlled. Examples of other biodegradable polymers include, but arenot limited to, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the modified mRNA inliposomes or microemulsions which are compatible with body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be used for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Properties of the Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of the following properties:

Bioavailability

The modified nucleic acid molecules and mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in bioavailability as compared to a composition lacking adelivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof a modified nucleic acid molecule administered to a mammal.Bioavailability can be assessed by measuring the area under the curve(AUC) or the maximum serum or plasma concentration (C_(max)) of theunchanged form of a compound following administration of the compound toa mammal. AUC is a determination of the area under the curve plottingthe serum or plasma concentration of a compound along the ordinate(Y-axis) against time along the abscissa (X-axis). Generally, the AUCfor a particular compound can be calculated using methods known to thoseof ordinary skill in the art and as described in G. S. Banker, ModernPharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, MarcelDekker, New York, Inc., 1996, herein incorporated by reference in itsentirety.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first modified nucleic acid molecule, measured as AUC, C_(max), orC_(min) in a mammal is greater, when co-administered with a deliveryagent as described herein, than when such co-administration does nottake place. In some embodiments, the bioavailability of the modifiednucleic acid molecule can increase by at least about 2%, at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100%.

Therapeutic Window

The modified nucleic acid molecules and mmRNA when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in the therapeutic window of the administered modified nucleicacid molecule composition as compared to the therapeutic window of theadministered modified nucleic acid molecule composition lacking adelivery agent as described herein. As used herein “therapeutic window”refers to the range of plasma concentrations, or the range of levels oftherapeutically active substance at the site of action, with a highprobability of eliciting a therapeutic effect. In some embodiments, thetherapeutic window of the modified nucleic acid molecule whenco-administered with a delivery agent as described herein can increaseby at least about 2%, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%.

Volume of Distribution

The modified nucleic acid molecules, when formulated into a compositionwith a delivery agent as described herein, can exhibit an improvedvolume of distribution (V_(dist)), e.g., reduced or targeted, relativeto a modified nucleic acid molecule composition lacking a delivery agentas described herein. The volume of distribution (V_(dist)) relates theamount of the drug in the body to the concentration of the drug in theblood or plasma. As used herein, the term “volume of distribution”refers to the fluid volume that would be required to contain the totalamount of the drug in the body at the same concentration as in the bloodor plasma: V_(dist) equals the amount of drug in the body/concentrationof drug in blood or plasma. For example, for a 10 mg dose and a plasmaconcentration of 10 mg/L, the volume of distribution would be 1 liter.The volume of distribution reflects the extent to which the drug ispresent in the extravascular tissue. A large volume of distributionreflects the tendency of a compound to bind to the tissue componentscompared with plasma protein binding. In a clinical setting, V_(dist)can be used to determine a loading dose to achieve a steady stateconcentration. In some embodiments, the volume of distribution of themodified nucleic acid molecule when co-administered with a deliveryagent as described herein can decrease at least about 2%, at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The protein expression may be determined from analyzing abiological sample collected from a mammal administered the modified mRNAof the present invention. In one embodiment, the expression proteinencoded by the modified mRNA administered to the mammal of at least 50pg/ml may be preferred. For example, a protein expression of 50-200pg/ml for the protein encoded by the modified mRNA delivered to themammal may be seen as a therapeutically effective amount of protein inthe mammal.

Detection of Modified Nucleic Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290; each of which areherein incorporated by reference in its entirety). Unlike untargetedmass spectrometry frequently used in biomarker discovery studies,targeted MS methods are peptide sequence-based modes of MS that focusthe full analytical capacity of the instrument on tens to hundreds ofselected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; hereinincorporated by reference in its entirety). The ionic species containedin solution may be transferred into the gas phase by dispersing a finespray of charge droplets, evaporating the solvent and ejecting the ionsfrom the charged droplets to generate a mist of highly charged droplets.The mist of highly charged droplets may be analyzed using at least 1, atleast 2, at least 3 or at least 4 mass analyzers such as, but notlimited to, a quadropole mass analyzer. Further, the mass spectrometrymethod may include a purification step. As a non-limiting example, thefirst quadrapole may be set to select a single m/z ratio so it mayfilter out other molecular ions having a different m/z ratio which mayeliminate complicated and time-consuming sample purification proceduresprior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894; herein incorporatedby reference in its entirety). As non-limiting examples, mass analyzersused in the MALDI analysis may include a linear time-of-flight (TOF), aTOF reflectron or a Fourier transform mass analyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

Uses of Modified Nucleic Acid Molecules Therapeutic Agents

The modified nucleic acid molecules and the proteins translated from themodified nucleic acid molecules described herein can be used astherapeutic agents. For example, a modified nucleic acid moleculedescribed herein can be administered to a subject, wherein the modifiednucleic acid molecule is translated in vivo to produce a therapeuticpeptide in the subject. Accordingly, provided herein are compositions,methods, kits, and reagents for treatment or prevention of disease orconditions in humans and other mammals. The active therapeutic agents ofthe present disclosure include, but are not limited to, modified nucleicacid molecules, cells containing modified nucleic acid molecules orpolypeptides translated from the modified nucleic acid molecules,polypeptides translated from modified nucleic acid molecules, and cellscontacted with cells containing modified nucleic acid molecules orpolypeptides translated from the modified nucleic acid molecules.

In certain embodiments, combination therapeutics are provided which maycontaining one or more modified nucleic acid molecules containingtranslatable regions along with a protein that inducesantibody-dependent cellular toxicity. As used herein “translatableregions” encode for a protein or proteins that may boost a subject'simmunity. For example, provided herein are therapeutics containing oneor more nucleic acids that encode trastuzumab and granulocyte-colonystimulating factor (G-CSF). In particular, such combination therapeuticsmay be useful in Her2+ breast cancer patients who develop inducedresistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy.2(6):795-8 (2010); herein incorporated by reference in its entirety).

Methods of inducing translation of a recombinant polypeptide in a cellpopulation using the modified nucleic acid molecules described hereinare also provided. Such translation can be in vivo, ex vivo, in culture,or in vitro. The cell population may be contacted with an effectiveamount of a composition containing a nucleic acid that has at least onenucleoside modification, and a translatable region encoding therecombinant polypeptide. The population may be contacted underconditions such that the nucleic acid may be localized into one or morecells of the cell population and the recombinant polypeptide may betranslated in the cell from the nucleic acid.

An effective amount of the composition may be provided based, at leastin part, on the target tissue, target cell type, means ofadministration, physical characteristics of the nucleic acid (e.g.,size, and extent of modified nucleosides), and other determinants. Ingeneral, an effective amount of the composition provides efficientprotein production in the cell, preferably more efficient than acomposition containing a corresponding unmodified nucleic acid molecule.Increased efficiency may be demonstrated by increased cell transfection(i.e., the percentage of cells transfected with the nucleic acid),increased protein translation from the nucleic acid, decreased nucleicacid degradation (as demonstrated, e.g., by increased duration ofprotein translation from a modified nucleic acid molecule), or reducedinnate immune response of the host cell.

Aspects of the present disclosure are directed to methods of inducing invivo translation of a recombinant polypeptide in a mammalian subject inneed thereof. Therein, an effective amount of a composition containing anucleic acid that has at least one nucleoside modification and atranslatable region encoding the recombinant polypeptide may beadministered to the subject using the delivery methods described herein.The nucleic acid may be provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide may be translated in the cell from thenucleic acid. The cell in which the nucleic acid is localized, or thetissue in which the cell is present, may be targeted with one or morethan one rounds of nucleic acid administration.

Other aspects of the present disclosure relate to transplantation ofcells containing modified nucleic acid molecules to a mammalian subject.Administration of cells to mammalian subjects is known to those ofordinary skill in the art, and include, but is not limited to, localimplantation (e.g., topical or subcutaneous administration), organdelivery or systemic injection (e.g., intravenous injection orinhalation), and the formulation of cells in pharmaceutically acceptablecarrier. Compositions containing modified nucleic acid molecules areformulated for administration intramuscularly, transarterially,intraperitoneally, intravenously, intranasally, subcutaneously,endoscopically, transdermally, or intrathecally. In some embodiments,the composition may be formulated for extended release.

The subject to whom the therapeutic agent may be administered suffersfrom or may be at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

In certain embodiments, the administered modified nucleic acid moleculedirects production of one or more recombinant polypeptides that providea functional activity which may be substantially absent in the cell inwhich the recombinant polypeptide may be translated. For example, themissing functional activity may be enzymatic, structural, or generegulatory in nature.

In other embodiments, the administration of a modified nucleic acidmolecule directs production of one or more recombinant polypeptides thatreplace a polypeptide (or multiple polypeptides) that may besubstantially absent in the cell in which the recombinant polypeptidemay be translated. Such absence may be due to a genetic mutation of theencoding gene or a regulatory pathway thereof. Alternatively, therecombinant polypeptide functions to antagonize the activity of anendogenous protein present in, on the surface of, or secreted from thecell. Usually, the activity of the endogenous protein may be deleteriousto the subject, for example, due to the mutation of the endogenousprotein resulting in altered activity or localization. Additionally, therecombinant polypeptide antagonizes, directly or indirectly, theactivity of a biological moiety present in, on the surface of, orsecreted from the cell. Examples of antagonized biological moietiesinclude, but are not limited to, lipids (e.g., cholesterol), alipoprotein (e.g., low density lipoprotein), a nucleic acid, acarbohydrate, or a small molecule toxin.

The recombinant proteins described herein may be engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

As described herein, a useful feature of the modified nucleic acidmolecules of the present disclosure is the capacity to reduce the innateimmune response of a cell to an exogenous nucleic acid. Provided aremethods for performing the titration, reduction or elimination of theimmune response in a cell or a population of cells. In some embodiments,the cell may be contacted with a first composition that contains a firstdose of a first exogenous nucleic acid including a translatable regionand at least one nucleoside modification, and the level of the innateimmune response of the cell to the first exogenous nucleic acid may bedetermined. Subsequently, the cell may be contacted with a secondcomposition, which includes a second dose of the first exogenous nucleicacid, the second dose containing a lesser amount of the first exogenousnucleic acid as compared to the first dose. Alternatively, the cell maybe contacted with a first dose of a second exogenous nucleic acid. Thesecond exogenous nucleic acid may contain one or more modifiednucleosides, which may be the same or different from the first exogenousnucleic acid or, alternatively, the second exogenous nucleic acid maynot contain modified nucleosides. The steps of contacting the cell withthe first composition and/or the second composition may be repeated oneor more times. Additionally, efficiency of protein production (e.g.,protein translation) in the cell may be optionally determined, and thecell may be re-transfected with the first and/or second compositionrepeatedly until a target protein production efficiency is achieved.

Therapeutics for Diseases and Conditions

Provided herein are methods for treating or preventing a symptom ofdiseases, characterized by missing or aberrant protein activity, bysupplying the missing protein activity or overcoming the aberrantprotein activity. Because of the rapid initiation of protein productionfollowing introduction of modified mRNA, as compared to viral DNAvectors, the compounds of the present disclosure are particularlyadvantageous in treating acute diseases such as sepsis, stroke, andmyocardial infarction. Moreover, an accurate titration of protein may beachievable using the modified mRNA of the present disclosure as themodified mRNA may be able to alter transcription rates and thus causechanges in gene expression.

Diseases characterized by dysfunctional or aberrant protein activityinclude, but are not limited to, cancer and proliferative diseases,genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes,neurodegenerative diseases, cardiovascular diseases, and metabolicdiseases. The present disclosure provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the modified nucleic acid moleculesprovided herein, wherein the modified nucleic acid molecules encode fora protein that antagonizes or otherwise overcomes the aberrant proteinactivity present in the cell of the subject. Specific examples of adysfunctional protein include, but are not limited to, the missensemutation variants of the cystic fibrosis transmembrane conductanceregulator (CFTR) gene, which produce a dysfunctional protein variant ofCFTR protein, which causes cystic fibrosis.

Multiple diseases may be characterized by missing (or substantiallydiminished such that proper protein function does not occur) proteinactivity. Such proteins may not be present, or they may be essentiallynon-functional.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a modified nucleic acidmolecule having a translatable region that encodes a functional CFTRpolypeptide, under conditions such that an effective amount of the CTFRpolypeptide is present in the cell. Preferred target cells areepithelial cells, such as the lung, and methods of administration aredetermined in view of the target tissue; i.e., for lung delivery, theRNA molecules are formulated for administration by inhalation.

In another embodiment, the present disclosure provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with a modified mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721; herein incorporated by reference in its entirety).

Methods of Cellular Nucleic Acid Delivery

Methods of the present disclosure enhance nucleic acid delivery into acell population, in vivo, ex vivo, or in culture. For example, a cellculture containing a plurality of host cells (e.g., eukaryotic cellssuch as yeast or mammalian cells) may be contacted with a compositionthat contains an modified nucleic acid molecule having at least onenucleoside modification and, optionally, a translatable region. Thecomposition may also generally contain a transfection reagent or othercompound that may increases the efficiency of modified nucleic acidmolecule uptake into the host cells. The modified nucleic acid moleculemay exhibit enhanced retention in the cell population, relative to acorresponding unmodified nucleic acid molecule. The retention of themodified nucleic acid molecule may greater than the retention of theunmodified nucleic acid molecule. In some embodiments, it is at leastabout 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greaterthan the retention of the unmodified nucleic acid molecule. Suchretention advantage may be achieved by one round of transfection withthe modified nucleic acid molecule, or may be obtained followingrepeated rounds of transfection.

In some embodiments, the modified nucleic acid molecule may be deliveredto a target cell population with one or more additional nucleic acids.Such delivery may be at the same time, or the modified nucleic acidmolecule is delivered prior to delivery of the one or more additionalnucleic acids. The additional one or more nucleic acids may be modifiednucleic acid molecules or unmodified nucleic acid molecules. It isunderstood that the initial presence of the modified nucleic acidmolecules may not substantially induce an innate immune response of thecell population and, moreover, that the innate immune response may notbe activated by the later presence of the unmodified nucleic acidmolecules. In this regard, the enhanced nucleic acid may not itselfcontain a translatable region, if the protein desired to be present inthe target cell population is translated from the unmodified nucleicacid molecules.

Targeting Moieties

In some embodiments, modified nucleic acid molecules are provided toexpress a protein-binding partner or a receptor on the surface of thecell, which may function to target the cell to a specific tissue spaceor to interact with a specific moiety, either in vivo or in vitro.Suitable protein-binding partners include, but are not limited to,antibodies and functional fragments thereof, scaffold proteins, orpeptides. Additionally, modified nucleic acid molecules may be employedto direct the synthesis and extracellular localization of lipids,carbohydrates, or other biological moieties.

Permanent Gene Expression Silencing

A method for epigenetically silencing gene expression in a mammaliansubject, comprising a nucleic acid where the translatable region encodesa polypeptide or polypeptides capable of directing sequence-specifichistone H3 methylation to initiate heterochromatin formation and reducegene transcription around specific genes for the purpose of silencingthe gene. For example, a gain-of-function mutation in the Janus Kinase 2gene is responsible for the family of Myeloproliferative Diseases.

Expression of Ligand or Receptor on Cell Surface

In some aspects and embodiments of the aspects described herein, themodified RNA can be used to express a ligand or ligand receptor on thesurface of a cell (e.g., a homing moiety). A ligand or ligand receptormoiety attached to a cell surface can permit the cell to have a desiredbiological interaction with a tissue or an agent in vivo. A ligand canbe an antibody, an antibody fragment, an aptamer, a peptide, a vitamin,a carbohydrate, a protein or polypeptide, a receptor, e.g., cell-surfacereceptor, an adhesion molecule, a glycoprotein, a sugar residue, atherapeutic agent, a drug, a glycosaminoglycan, or any combinationthereof. For example, a ligand can be an antibody that recognizes acancer-cell specific antigen, rendering the cell capable ofpreferentially interacting with tumor cells to permit tumor-specificlocalization of a modified cell. A ligand can confer the ability of acell composition to accumulate in a tissue to be treated, since apreferred ligand may be capable of interacting with a target molecule onthe external face of a tissue to be treated. Ligands having limitedcross-reactivity to other tissues are generally preferred.

In some cases, a ligand can act as a homing moiety which permits thecell to target to a specific tissue or interact with a specific ligand.Such homing moieties can include, but are not limited to, any member ofa specific binding pair, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((SCFV)2 fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other homing moieties include forexample, aptamers, receptors, and fusion proteins.

In some embodiments, the homing moiety may be a surface-bound antibody,which can permit tuning of cell targeting specificity. This isespecially useful since highly specific antibodies can be raised againstan epitope of interest for the desired targeting site. In oneembodiment, multiple antibodies are expressed on the surface of a cell,and each antibody can have a different specificity for a desired target.Such approaches can increase the avidity and specificity of hominginteractions.

A skilled artisan can select any homing moiety based on the desiredlocalization or function of the cell, for example an estrogen receptorligand, such as tamoxifen, can target cells to estrogen-dependent breastcancer cells that have an increased number of estrogen receptors on thecell surface. Other non-limiting examples of ligand/receptorinteractions include CCRI (e.g., for treatment of inflamed joint tissuesor brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8(e.g., targeting to lymph node tissue), CCR6, CCR9, CCR10 (e.g., totarget to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., fortreatment of inflammation and inflammatory disorders, bone marrow),Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4NCAM-1 (e.g.,targeting to endothelium). In general, any receptor involved intargeting (e.g., cancer metastasis) can be harnessed for use in themethods and compositions described herein.

Mediators of Cell Death

In one embodiment, a modified nucleic acid molecule composition can beused to induce apoptosis in a cell (e.g., a cancer cell) by increasingthe expression of a death receptor, a death receptor ligand or acombination thereof. This method can be used to induce cell death in anydesired cell and has particular usefulness in the treatment of cancerwhere cells escape natural apoptotic signals.

Apoptosis can be induced by multiple independent signaling pathways thatconverge upon a final effector mechanism consisting of multipleinteractions between several “death receptors” and their ligands, whichbelong to the tumor necrosis factor (TNF) receptor/ligand superfamily.The best-characterized death receptors are CD95 (“Fas”), TNFRI (p55),death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). Thefinal effector mechanism of apoptosis may be the activation of a seriesof proteinases designated as caspases. The activation of these caspasesresults in the cleavage of a series of vital cellular proteins and celldeath. The molecular mechanism of death receptors/ligands-inducedapoptosis is well known in the art. For example, Fas/FasL-mediatedapoptosis is induced by binding of three FasL molecules which inducestrimerization of Fas receptor via C-terminus death domains (DDs), whichin turn recruits an adapter protein FADD (Fas-associated protein withdeath domain) and Caspase-8. The oligomerization of this trimolecularcomplex, Fas/FAIDD/caspase-8, results in proteolytic cleavage ofproenzyme caspase-8 into active caspase-8 that, in turn, initiates theapoptosis process by activating other downstream caspases throughproteolysis, including caspase-3. Death ligands in general are apoptoticwhen formed into trimers or higher order of structures. As monomers,they may serve as antiapoptotic agents by competing with the trimers forbinding to the death receptors.

In one embodiment, the modified nucleic acid molecule compositionencodes for a death receptor (e.g., Fas, TRAIL, TRAMO, TNFR, TLR etc).Cells made to express a death receptor by transfection of modified RNAbecome susceptible to death induced by the ligand that activates thatreceptor. Similarly, cells made to express a death ligand, e.g., ontheir surface, will induce death of cells with the receptor when thetransfected cell contacts the target cell. In another embodiment, themodified RNA composition encodes for a death receptor ligand (e.g.,FasL, TNF, etc). In another embodiment, the modified RNA compositionencodes a caspase (e.g., caspase 3, caspase 8, caspase 9 etc). Wherecancer cells often exhibit a failure to properly differentiate to anon-proliferative or controlled proliferative form, in anotherembodiment, the synthetic, modified RNA composition encodes for both adeath receptor and its appropriate activating ligand. In anotherembodiment, the synthetic, modified RNA composition encodes for adifferentiation factor that when expressed in the cancer cell, such as acancer stem cell, will induce the cell to differentiate to anon-pathogenic or nonself-renewing phenotype (e.g., reduced cell growthrate, reduced cell division etc) or to induce the cell to enter adormant cell phase (e.g., G₀ resting phase).

One of skill in the art will appreciate that the use ofapoptosis-inducing techniques may require that the modified nucleic acidmolecules are appropriately targeted to e.g., tumor cells to preventunwanted wide-spread cell death. Thus, one can use a delivery mechanism(e.g., attached ligand or antibody, targeted liposome etc) thatrecognizes a cancer antigen such that the modified nucleic acidmolecules are expressed only in cancer cells.

Kits and Devices Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits for proteinproduction, comprising a first modified nucleic acid molecule or mmRNAcomprising a translatable region. The kit may further comprise packagingand instructions and/or a delivery agent to form a formulationcomposition. The delivery agent may comprise a saline, a bufferedsolution, a lipidoid or any delivery agent disclosed herein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See e.g., U.S. Pub. No.20120258046; herein incorporated by reference in its entirety). In afurther embodiment, the buffer solutions may be precipitated or it maybe lyophilized. The amount of each component may be varied to enableconsistent, reproducible higher concentration saline or simple bufferformulations. The components may also be varied in order to increase thestability of modified nucleic acid molecules and mmRNA in the buffersolution over a period of time and/or under a variety of conditions.

In one aspect, the present invention provides kits for proteinproduction, comprising: a modified nucleic acid molecule or mmRNAcomprising a translatable region, provided in an amount effective toproduce a desired amount of a protein encoded by the translatable regionwhen introduced into a target cell; a second modified nucleic acidmolecule or mmRNA comprising an inhibitory nucleic acid, provided in anamount effective to substantially inhibit the innate immune response ofthe cell; and packaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a modified nucleic acid molecule or mmRNAcomprising a translatable region, wherein the nucleic acid exhibitsreduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a modified nucleic acid molecule or mmRNAcomprising a translatable region, wherein the nucleic acid exhibitsreduced degradation by a cellular nuclease, and a mammalian cellsuitable for translation of the translatable region of the first nucleicacid.

Devices

The present invention provides for devices which may incorporatemodified nucleic acid molecules or mmRNA that encode polypeptides ofinterest. These devices contain in a stable formulation the reagents tosynthesize a nucleic acid in a formulation available to be immediatelydelivered to a subject in need thereof, such as a human patient.Non-limiting examples of such a polypeptide of interest include a growthfactor and/or angiogenesis stimulator for wound healing, a peptideantibiotic to facilitate infection control, and an antigen to rapidlystimulate an immune response to a newly identified virus.

In some embodiments the device is self-contained, and is optionallycapable of wireless remote access to obtain instructions for synthesisand/or analysis of the generated modified nucleic acid molecule ormmRNA. The device is capable of mobile synthesis of at least onemodified nucleic acid molecule or mmRNA and preferably an unlimitednumber of different modified nucleic acid molecules or mmRNA. In certainembodiments, the device is capable of being transported by one or asmall number of individuals. In other embodiments, the device is scaledto fit on a benchtop or desk. In other embodiments, the device is scaledto fit into a suitcase, backpack or similarly sized object.

In another embodiment, the device may be a point of care or handhelddevice. In further embodiments, the device is scaled to fit into avehicle, such as a car, truck or ambulance, or a military vehicle suchas a tank or personnel carrier. The information necessary to generate amodified mRNA encoding polypeptide of interest is present within acomputer readable medium present in the device.

In one embodiment, a device may be used to assess levels of a proteinwhich has been administered in the form of a modified nucleic acid ormmRNA. The device may comprise a blood, urine or other biofluidic test.

In some embodiments, the device is capable of communication (e.g.,wireless communication) with a database of nucleic acid and polypeptidesequences. The device contains at least one sample block for insertionof one or more sample vessels. Such sample vessels are capable ofaccepting in liquid or other form any number of materials such astemplate DNA, nucleotides, enzymes, buffers, and other reagents. Thesample vessels are also capable of being heated and cooled by contactwith the sample block. The sample block is generally in communicationwith a device base with one or more electronic control units for the atleast one sample block. The sample block preferably contains a heatingmodule, such heating molecule capable of heating and/or cooling thesample vessels and contents thereof to temperatures between about −20 Cand above +100 C. The device base is in communication with a voltagesupply such as a battery or external voltage supply. The device alsocontains means for storing and distributing the materials for RNAsynthesis.

Optionally, the sample block contains a module for separating thesynthesized nucleic acids. Alternatively, the device contains aseparation module operably linked to the sample block. Preferably thedevice contains a means for analysis of the synthesized nucleic acid.Such analysis includes sequence identity (demonstrated such as byhybridization), absence of non-desired sequences, measurement ofintegrity of synthesized mRNA (such has by microfluidic viscometrycombined with spectrophotometry), and concentration and/or potency ofmodified RNA (such as by spectrophotometry).

In certain embodiments, the device is combined with a means fordetection of pathogens present in a biological material obtained from asubject, e.g., the IBIS PLEX-ID system (Abbott, Abbott Park, Ill.) formicrobial identification.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662; each of which is hereinincorporated by reference in their entirety. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 (herein incorporated by reference in itsentirety) and functional equivalents thereof. Jet injection deviceswhich deliver liquid compositions to the dermis via a liquid jetinjector and/or via a needle which pierces the stratum corneum andproduces a jet which reaches the dermis are suitable. Jet injectiondevices are described, for example, in U.S. Pat. Nos. 5,480,381;5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; andPCT publications WO 97/37705 and WO 97/13537; each of which are hereinincorporated by reference in their entirety. Ballistic powder/particledelivery devices which use compressed gas to accelerate vaccine inpowder form through the outer layers of the skin to the dermis aresuitable. Alternatively or additionally, conventional syringes may beused in the classical mantoux method of intradermal administration.

In some embodiments, the device may be a pump or comprise a catheter foradministration of compounds or compositions of the invention across theblood brain barrier. Such devices include but are not limited to apressurized olfactory delivery device, iontophoresis devices,multi-layered microfluidic devices, and the like. Such devices may beportable or stationary. They may be implantable or externally tetheredto the body or combinations thereof.

Devices for administration may be employed to deliver the modifiednucleic acid molecules or mmRNA of the present invention according tosingle, multi- or split-dosing regimens taught herein. Such devices aredescribed below.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicesmay be utilized to deliver the single, multi- or split dosescontemplated herein.

A method for delivering therapeutic agents to a solid tissue has beendescribed by Bahrami et al. and is taught for example in US PatentPublication 20110230839, the contents of which are incorporated hereinby reference in their entirety. According to Bahrami, an array ofneedles is incorporated into a device which delivers a substantiallyequal amount of fluid at any location in said solid tissue along eachneedle's length.

A device for delivery of biological material across the biologicaltissue has been described by Kodgule et al. and is taught for example inUS Patent Publication 20110172610, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple hollow micro-needles made of one or more metals andhaving outer diameters from about 200 microns to about 350 microns andlengths of at least 100 microns are incorporated into the device whichdelivers peptides, proteins, carbohydrates, nucleic acid molecules,lipids and other pharmaceutically active ingredients or combinationsthereof.

A delivery probe for delivering a therapeutic agent to a tissue has beendescribed by Gunday et al. and is taught for example in US PatentPublication 20110270184, the contents of each of which are incorporatedherein by reference in their entirety. According to Gunday, multipleneedles are incorporated into the device which moves the attachedcapsules between an activated position and an inactivated position toforce the agent out of the capsules through the needles.

A multiple-injection medical apparatus has been described by Assaf andis taught for example in US Patent Publication 20110218497, the contentsof which are incorporated herein by reference in their entirety.According to Assaf, multiple needles are incorporated into the devicewhich has a chamber connected to one or more of said needles and a meansfor continuously refilling the chamber with the medical fluid after eachinjection.

In one embodiment, the modified nucleic acid molecule or mmRNA isadministered subcutaneously or intramuscularly via at least 3 needles tothree different, optionally adjacent, sites simultaneously, or within a60 minutes period (e.g., administration to 4,5, 6, 7, 8, 9, or 10 sitessimultaneously or within a 60 minute period). The split doses can beadministered simultaneously to adjacent tissue using the devicesdescribed in U.S. Patent Publication Nos. 20110230839 and 20110218497,each of which is incorporated herein by reference in their entirety.

An at least partially implantable system for injecting a substance intoa patient's body, in particular a penis erection stimulation system hasbeen described by Forsell and is taught for example in US PatentPublication 20110196198, the contents of which are incorporated hereinby reference in their entirety. According to Forsell, multiple needlesare incorporated into the device which is implanted along with one ormore housings adjacent the patient's left and right corpora cavernosa. Areservoir and a pump are also implanted to supply drugs through theneedles.

A method for the transdermal delivery of a therapeutic effective amountof iron has been described by Berenson and is taught for example in USPatent Publication 20100130910, the contents of which are incorporatedherein by reference in their entirety. According to Berenson, multipleneedles may be used to create multiple micro channels in stratum corneumto enhance transdermal delivery of the ionic iron on an iontophoreticpatch.

A method for delivery of biological material across the biologicaltissue has been described by Kodgule et al and is taught for example inUS Patent Publication 20110196308, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple biodegradable microneedles containing a therapeuticactive ingredient are incorporated in a device which delivers proteins,carbohydrates, nucleic acid molecules, lipids and other pharmaceuticallyactive ingredients or combinations thereof.

A transdermal patch comprising a botulinum toxin composition has beendescribed by Donovan and is taught for example in US Patent Publication20080220020, the contents of which are incorporated herein by referencein their entirety. According to Donovan, multiple needles areincorporated into the patch which delivers botulinum toxin under stratumcorneum through said needles which project through the stratum corneumof the skin without rupturing a blood vessel.

A small, disposable drug reservoir, or patch pump, which can holdapproximately 0.2 to 15 mL of liquid formulations can be placed on theskin and deliver the formulation continuously subcutaneously using asmall bore needed (e.g., 26 to 34 gauge). As non-limiting examples, thepatch pump may be 50 mm by 76 mm by 20 mm spring loaded having a 30 to34 gauge needle (BD™ Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mmby 17 mm with a 2 mL reservoir used for drug delivery such as insulin(OMNIPOD®, Insulet Corporation Bedford, Mass.), or 43-60 mm diameter, 10mm thick with a 0.5 to 10 mL reservoir (PATCHPUMP®, SteadyMedTherapeutics, San Francisco, Calif.). Further, the patch pump may bebattery powered and/or rechargeable.

A cryoprobe for administration of an active agent to a location ofcryogenic treatment has been described by Toubia and is taught forexample in US Patent Publication 20080140061, the contents of which areincorporated herein by reference in their entirety. According to Toubia,multiple needles are incorporated into the probe which receives theactive agent into a chamber and administers the agent to the tissue.

A method for treating or preventing inflammation or promoting healthyjoints has been described by Stock et al and is taught for example in USPatent Publication 20090155186, the contents of which are incorporatedherein by reference in their entirety. According to Stock, multipleneedles are incorporated in a device which administers compositionscontaining signal transduction modulator compounds.

A multi-site injection system has been described by Kimmell et al. andis taught for example in US Patent Publication 20100256594, the contentsof which are incorporated herein by reference in their entirety.According to Kimmell, multiple needles are incorporated into a devicewhich delivers a medication into a stratum corneum through the needles.

A method for delivering interferons to the intradermal compartment hasbeen described by Dekker et al. and is taught for example in US PatentPublication 20050181033, the contents of which are incorporated hereinby reference in their entirety. According to Dekker, multiple needleshaving an outlet with an exposed height between 0 and 1 mm areincorporated into a device which improves pharmacokinetics andbioavailability by delivering the substance at a depth between 0.3 mmand 2 mm.

A method for delivering genes, enzymes and biological agents to tissuecells has described by Desai and is taught for example in US PatentPublication 20030073908, the contents of which are incorporated hereinby reference in their entirety. According to Desai, multiple needles areincorporated into a device which is inserted into a body and delivers amedication fluid through said needles.

A method for treating cardiac arrhythmias with fibroblast cells has beendescribed by Lee et al and is taught for example in US PatentPublication 20040005295, the contents of which are incorporated hereinby reference in their entirety. According to Lee, multiple needles areincorporated into the device which delivers fibroblast cells into thelocal region of the tissue.

A method using a magnetically controlled pump for treating a brain tumorhas been described by Shachar et al. and is taught for example in U.S.Pat. No. 7,799,012 (method) and 7,799,016 (device), the contents ofwhich are incorporated herein by reference in their entirety. AccordingShachar, multiple needles were incorporated into the pump which pushes amedicating agent through the needles at a controlled rate.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al. and are taught for examplein U.S. Pat. No. 8,029,496, the contents of which are incorporatedherein by reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A micro-needle transdermal transport device has been described by Angelet al and is taught for example in U.S. Pat. No. 7,364,568, the contentsof which are incorporated herein by reference in their entirety.According to Angel, multiple needles are incorporated into the devicewhich transports a substance into a body surface through the needleswhich are inserted into the surface from different directions. Themicro-needle transdermal transport device may be a solid micro-needlesystem or a hollow micro-needle system. As a non-limiting example, thesolid micro-needle system may have up to a 0.5 mg capacity, with300-1500 solid micro-needles per cm² about 150-700 μm tall coated with adrug. The micro-needles penetrate the stratum corneum and remain in theskin for short duration (e.g., 20 seconds to 15 minutes). In anotherexample, the hollow micro-needle system has up to a 3 mL capacity todeliver liquid formulations using 15-20 microneedles per cm2 beingapproximately 950 μm tall. The micro-needles penetrate the skin to allowthe liquid formulations to flow from the device into the skin. Thehollow micro-needle system may be worn from 1 to 30 minutes depending onthe formulation volume and viscocity.

A device for subcutaneous infusion has been described by Dalton et aland is taught for example in U.S. Pat. No. 7,150,726, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Dalton, multiple needles are incorporated into the device whichdelivers fluid through the needles into a subcutaneous tissue.

A device and a method for intradermal delivery of vaccines and genetherapeutic agents through microcannula have been described by Miksztaet al. and are taught for example in U.S. Pat. No. 7,473,247, thecontents of which are incorporated herein by reference in theirentirety. According to Mitszta, at least one hollow micro-needle isincorporated into the device which delivers the vaccines to thesubject's skin to a depth of between 0.025 mm and 2 mm.

A method of delivering insulin has been described by Pettis et al and istaught for example in U.S. Pat. No. 7,722,595, the contents of which areincorporated herein by reference in their entirety. According to Pettis,two needles are incorporated into a device wherein both needles insertessentially simultaneously into the skin with the first at a depth ofless than 2.5 mm to deliver insulin to intradermal compartment and thesecond at a depth of greater than 2.5 mm and less than 5.0 mm to deliverinsulin to subcutaneous compartment.

Cutaneous injection delivery under suction has been described byKochamba et al. and is taught for example in U.S. Pat. No. 6,896,666,the contents of which are incorporated herein by reference in theirentirety. According to Kochamba, multiple needles in relative adjacencywith each other are incorporated into a device which injects a fluidbelow the cutaneous layer.

A device for withdrawing or delivering a substance through the skin hasbeen described by Down et al and is taught for example in U.S. Pat. No.6,607,513, the contents of which are incorporated herein by reference intheir entirety. According to Down, multiple skin penetrating memberswhich are incorporated into the device have lengths of about 100 micronsto about 2000 microns and are about 30 to 50 gauge.

A device for delivering a substance to the skin has been described byPalmer et al and is taught for example in U.S. Pat. No. 6,537,242, thecontents of which are incorporated herein by reference in theirentirety. According to Palmer, an array of micro-needles is incorporatedinto the device which uses a stretching assembly to enhance the contactof the needles with the skin and provides a more uniform delivery of thesubstance.

A perfusion device for localized drug delivery has been described byZamoyski and is taught for example in U.S. Pat. No. 6,468,247, thecontents of which are incorporated herein by reference in theirentirety. According to Zamoyski, multiple hypodermic needles areincorporated into the device which injects the contents of thehypodermics into a tissue as said hypodermics are being retracted.

A method for enhanced transport of drugs and biological molecules acrosstissue by improving the interaction between micro-needles and human skinhas been described by Prausnitz et al. and is taught for example in U.S.Pat. No. 6,743,211, the contents of which are incorporated herein byreference in their entirety. According to Prausnitz, multiplemicro-needles are incorporated into a device which is able to present amore rigid and less deformable surface to which the micro-needles areapplied.

A device for intraorgan administration of medicinal agents has beendescribed by Ting et al and is taught for example in U.S. Pat. No.6,077,251, the contents of which are incorporated herein by reference intheir entirety. According to Ting, multiple needles having side openingsfor enhanced administration are incorporated into a device which byextending and retracting said needles from and into the needle chamberforces a medicinal agent from a reservoir into said needles and injectssaid medicinal agent into a target organ.

A multiple needle holder and a subcutaneous multiple channel infusionport has been described by Brown and is taught for example in U.S. Pat.No. 4,695,273, the contents of which are incorporated herein byreference in their entirety. According to Brown, multiple needles on theneedle holder are inserted through the septum of the infusion port andcommunicate with isolated chambers in said infusion port.

A dual hypodermic syringe has been described by Horn and is taught forexample in U.S. Pat. No. 3,552,394, the contents of which areincorporated herein by reference in their entirety. According to Horn,two needles incorporated into the device are spaced apart less than 68mm and may be of different styles and lengths, thus enabling injectionsto be made to different depths.

A syringe with multiple needles and multiple fluid compartments has beendescribed by Hershberg and is taught for example in U.S. Pat. No.3,572,336, the contents of which are incorporated herein by reference intheir entirety. According to Hershberg, multiple needles areincorporated into the syringe which has multiple fluid compartments andis capable of simultaneously administering incompatible drugs which arenot able to be mixed for one injection.

A surgical instrument for intradermal injection of fluids has beendescribed by Eliscu et al. and is taught for example in U.S. Pat. No.2,588,623, the contents of which are incorporated herein by reference intheir entirety. According to Eliscu, multiple needles are incorporatedinto the instrument which injects fluids intradermally with a widerdisperse.

An apparatus for simultaneous delivery of a substance to multiple breastmilk ducts has been described by Hung and is taught for example in EP1818017, the contents of which are incorporated herein by reference intheir entirety. According to Hung, multiple lumens are incorporated intothe device which inserts though the orifices of the ductal networks anddelivers a fluid to the ductal networks.

A catheter for introduction of medications to the tissue of a heart orother organs has been described by Tkebuchava and is taught for examplein WO2006138109, the contents of which are incorporated herein byreference in their entirety. According to Tkebuchava, two curved needlesare incorporated which enter the organ wall in a flattened trajectory.

Devices for delivering medical agents have been described by Mckay etal. and are taught for example in WO2006118804, the content of which areincorporated herein by reference in their entirety. According to Mckay,multiple needles with multiple orifices on each needle are incorporatedinto the devices to facilitate regional delivery to a tissue, such asthe interior disc space of a spinal disc.

A method for directly delivering an immunomodulatory substance into anintradermal space within a mammalian skin has been described by Pettisand is taught for example in WO2004020014, the contents of which areincorporated herein by reference in their entirety. According to Pettis,multiple needles are incorporated into a device which delivers thesubstance through the needles to a depth between 0.3 mm and 2 mm.

Methods and devices for administration of substances into at least twocompartments in skin for systemic absorption and improvedpharmacokinetics have been described by Pettis et al. and are taught forexample in WO2003094995, the contents of which are incorporated hereinby reference in their entirety. According to Pettis, multiple needleshaving lengths between about 300 μm and about 5 mm are incorporated intoa device which delivers to intradermal and subcutaneous tissuecompartments simultaneously.

A drug delivery device with needles and a roller has been described byZimmerman et al. and is taught for example in WO2012006259, the contentsof which are incorporated herein by reference in their entirety.According to Zimmerman, multiple hollow needles positioned in a rollerare incorporated into the device which delivers the content in areservoir through the needles as the roller rotates.

A drug delivery device such as a stent is known in the art and is taughtfor example in U.S. Pub. Nos. US20060020329, US20040172127 andUS20100161032; the contents of which are herein incorporated byreference in their entirety. Formulations of the modified nucleic acidmolecules and mmRNA described herein may be delivered using stents.Additionally, stents used herein may be able to deliver multiplemodified nucleic acid molecules and/or formulations at the same orvaried rates of delivery. Non-limiting examples of manufacturers ofstents include CORDIS® (Miami, Fla.) (CYPHER®), Boston ScientificCorporation (Natick, Mass.) (TAXUS®), Medtronic (Minneapolis, Minn.)(ENDEAVOUR®) and Abbott (Abbott Park, Ill.) (XIENCE V®).

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens may be employed toadminister the mmRNA of the present invention on a single, multi- orsplit dosing schedule. Such methods and devices are described below.

A catheter-based delivery of skeletal myoblasts to the myocardium ofdamaged hearts has been described by Jacoby et al and is taught forexample in US Patent Publication 20060263338, the contents of which areincorporated herein by reference in their entirety. According to Jacoby,multiple needles are incorporated into the device at least part of whichis inserted into a blood vessel and delivers the cell compositionthrough the needles into the localized region of the subject's heart.

An apparatus for treating asthma using neurotoxin has been described byDeem et al and is taught for example in US Patent Publication20060225742, the contents of which are incorporated herein by referencein their entirety. According to Deem, multiple needles are incorporatedinto the device which delivers neurotoxin through the needles into thebronchial tissue.

A method for administering multiple-component therapies has beendescribed by Nayak and is taught for example in U.S. Pat. No. 7,699,803,the contents of which are incorporated herein by reference in theirentirety. According to Nayak, multiple injection cannulas may beincorporated into a device wherein depth slots may be included forcontrolling the depth at which the therapeutic substance is deliveredwithin the tissue.

A surgical device for ablating a channel and delivering at least onetherapeutic agent into a desired region of the tissue has been describedby McIntyre et al and is taught for example in U.S. Pat. No. 8,012,096,the contents of which are incorporated herein by reference in theirentirety. According to McIntyre, multiple needles are incorporated intothe device which dispenses a therapeutic agent into a region of tissuesurrounding the channel and is particularly well suited fortransmyocardial revascularization operations.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al and are taught for example inU.S. Pat. No. 8,029,496, the contents of which are incorporated hereinby reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A device and a method for delivering fluid into a flexible biologicalbarrier have been described by Yeshurun et al. and are taught forexample in U.S. Pat. No. 7,998,119 (device) and 8,007,466 (method), thecontents of which are incorporated herein by reference in theirentirety. According to Yeshurun, the micro-needles on the devicepenetrate and extend into the flexible biological barrier and fluid isinjected through the bore of the hollow micro-needles.

A method for epicardially injecting a substance into an area of tissueof a heart having an epicardial surface and disposed within a torso hasbeen described by Bonner et al and is taught for example in U.S. Pat.No. 7,628,780, the contents of which are incorporated herein byreference in their entirety. According to Bonner, the devices haveelongate shafts and distal injection heads for driving needles intotissue and injecting medical agents into the tissue through the needles.

A device for sealing a puncture has been described by Nielsen et al andis taught for example in U.S. Pat. No. 7,972,358, the contents of whichare incorporated herein by reference in their entirety. According toNielsen, multiple needles are incorporated into the device whichdelivers a closure agent into the tissue surrounding the puncture tract.

A method for myogenesis and angiogenesis has been described by Chiu etal. and is taught for example in U.S. Pat. No. 6,551,338, the contentsof which are incorporated herein by reference in their entirety.According to Chiu, 5 to 15 needles having a maximum diameter of at least1.25 mm and a length effective to provide a puncture depth of 6 to 20 mmare incorporated into a device which inserts into proximity with amyocardium and supplies an exogeneous angiogenic or myogenic factor tosaid myocardium through the conduits which are in at least some of saidneedles.

A method for the treatment of prostate tissue has been described byBolmsj et al. and is taught for example in U.S. Pat. No. 6,524,270, thecontents of which are incorporated herein by reference in theirentirety. According to Bolmsj, a device comprising a catheter which isinserted through the urethra has at least one hollow tip extendible intothe surrounding prostate tissue. An astringent and analgesic medicine isadministered through said tip into said prostate tissue.

A method for infusing fluids to an intraosseous site has been describedby Findlay et al. and is taught for example in U.S. Pat. No. 6,761,726,the contents of which are incorporated herein by reference in theirentirety. According to Findlay, multiple needles are incorporated into adevice which is capable of penetrating a hard shell of material coveredby a layer of soft material and delivers a fluid at a predetermineddistance below said hard shell of material.

A device for injecting medications into a vessel wall has been describedby Vigil et al. and is taught for example in U.S. Pat. No. 5,713,863,the contents of which are incorporated herein by reference in theirentirety. According to Vigil, multiple injectors are mounted on each ofthe flexible tubes in the device which introduces a medication fluidthrough a multi-lumen catheter, into said flexible tubes and out of saidinjectors for infusion into the vessel wall.

A catheter for delivering therapeutic and/or diagnostic agents to thetissue surrounding a bodily passageway has been described by Faxon etal. and is taught for example in U.S. Pat. No. 5,464,395, the contentsof which are incorporated herein by reference in their entirety.According to Faxon, at least one needle cannula is incorporated into thecatheter which delivers the desired agents to the tissue through saidneedles which project outboard of the catheter.

Balloon catheters for delivering therapeutic agents have been describedby Orr and are taught for example in WO2010024871, the contents of whichare incorporated herein by reference in their entirety. According toOrr, multiple needles are incorporated into the devices which deliverthe therapeutic agents to different depths within the tissue. In anotheraspect, drug-eluting balloons may be used to deliver the formulationsdescribed herein. The drug-eluting balloons may be used in target lesionapplications such as, but are not limited to, in-stent restenosis,treating lesion in tortuous vessels, bifurcation lesions,femoral/popliteal lesions and below the knee lesions.

A device for deliverying therapeutic agents (e.g., modified nucleic acidmolecules or mmRNA) to tissue disposed about a lumin has been describedby Perry et al. and is taught for example in U.S. Pat. Pub.US20100125239, the contents of which are herein incorporated byreference in their entirety. According to Perry, the catheter has aballoon which may be coated with a therapeutic agent by methods known inthe art and described in Perry. When the balloon expands, thetherapeutic agent will contact the surrounding tissue. The device mayadditionally have a heat source to change the temperature of the coatingon the balloon to release the thereapeutic agent to the tissue.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current may be employed todeliver the mmRNA of the present invention according to the single,multi- or split dosing regimens taught herein. Such methods and devicesare described below.

An electro collagen induction therapy device has been described byMarquez and is taught for example in US Patent Publication 20090137945,the contents of which are incorporated herein by reference in theirentirety. According to Marquez, multiple needles are incorporated intothe device which repeatedly pierce the skin and draw in the skin aportion of the substance which is applied to the skin first.

An electrokinetic system has been described by Etheredge et al. and istaught for example in US Patent Publication 20070185432, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Etheredge, micro-needles are incorporated into a device which drivesby an electrical current the medication through the needles into thetargeted treatment site.

An iontophoresis device has been described by Matsumura et al. and istaught for example in U.S. Pat. No. 7,437,189, the contents of which areincorporated herein by reference in their entirety. According toMatsumura, multiple needles are incorporated into the device which iscapable of delivering ionizable drug into a living body at higher speedor with higher efficiency.

Intradermal delivery of biologically active agents by needle-freeinjection and electroporation has been described by Hoffmann et al andis taught for example in U.S. Pat. No. 7,171,264, the contents of whichare incorporated herein by reference in their entirety. According toHoffmann, one or more needle-free injectors are incorporated into anelectroporation device and the combination of needle-free injection andelectroporation is sufficient to introduce the agent into cells in skin,muscle or mucosa.

A method for electropermeabilization-mediated intracellular delivery hasbeen described by Lundkvist et al. and is taught for example in U.S.Pat. No. 6,625,486, the contents of which are incorporated herein byreference in their entirety. According to Lundkvist, a pair of needleelectrodes is incorporated into a catheter. Said catheter is positionedinto a body lumen followed by extending said needle electrodes topenetrate into the tissue surrounding said lumen. Then the deviceintroduces an agent through at least one of said needle electrodes andapplies electric field by said pair of needle electrodes to allow saidagent pass through the cell membranes into the cells at the treatmentsite.

A delivery system for transdermal immunization has been described byLevin et al. and is taught for example in WO2006003659, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Levin, multiple electrodes are incorporated into the device whichapplies electrical energy between the electrodes to generate microchannels in the skin to facilitate transdermal delivery.

A method for delivering RF energy into skin has been described bySchomacker and is taught for example in WO2011163264, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Schomacker, multiple needles are incorporated into a device whichapplies vacuum to draw skin into contact with a plate so that needlesinsert into skin through the holes on the plate and deliver RF energy.

DEFINITIONS

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agents(e.g., a modified nucleic acid or mmRNA encoding an anti-microbialpolypeptide (e.g., an anti-bacterial polypeptide), e.g., ananti-microbial polypeptide described herein and an anti-microbial agent(e.g., an anti-microbial polypeptide or a small molecule anti-microbialcompound described herein)) are administered to a subject at the sametime or within an interval such that there may be an overlap of aneffect of each agent on the patient. In some embodiments, they areadministered within about 60, 30, 15, 10, 5, or 1 minute of one another.In some embodiments, the administrations of the agents are spacedsufficiently close together such that a combinatorial (e.g., asynergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNA of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological affect on that organism,is considered to be biologically active. In particular embodiments, themodified nucleic acid or mmRNA of the present invention may beconsidered biologically active if even a portion of the modified nucleicacid or mmRNA is biologically active or mimics an activity consideredbiologically relevant.

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group and R^(N1) isas defined herein). Exemplary unsubstituted acylamino groups includefrom 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21,from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀ alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionallysubstituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplary unsubstitutedalkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₄₀ alkoxycarbonyl-C₁₋₁₀ alkyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). In some embodiments, each alkyl andalkoxy group is further independently substituted with 1, 2, 3, or 4substituents as described herein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉heterocyclyl)oxy; (8)hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), wherein each of these recited R^(N1) groups can beoptionally substituted, as defined herein for each group; or two R^(N1)combine to form a heterocyclyl or an N-protecting group, and whereineach R^(N2) is, independently, H, alkyl, or aryl. The amino groups ofthe invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N)2, SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(L′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula —OR,where R is a C₃₋₈ cycloalkyl group, as defined herein, unless otherwisespecified. The cycloalkyl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein. Exemplary unsubstitutedcycloalkoxy groups are from 3 to 8 carbons.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl,and the like. When the cycloalkyl group includes one carbon-carbondouble bond, the cycloalkyl group can be referred to as a “cycloalkenyl”group. Exemplary cycloalkenyl groups include cyclopentenyl,cyclohexenyl, and the like. The cycloalkyl groups of this invention canbe optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde);(2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3)C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diasteromer” means stereoisomers that are not mirror images ofone another and are non-superimposable.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂BOCH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where E′ is selected from the group consisting of —N— and —CH—; F′ isselected from the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—,—CH═N—, —CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—,—O—, and —S—; and G′ is selected from the group consisting of —CH— and—N—. Any of the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl)imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” ^(3rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “thiol” represents an —SH group.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone—enol pairs, amide—imidicacid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of a modifiednucleic acid or mmRNA to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dose splitting factor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least amodified nucleic acid or mmRNA and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form mmRNA multimers (e.g., throughlinkage of two or more modified nucleic acid molecules or mmRNAmolecules) or mmRNA conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers andderivatives thereof. Other examples include, but are not limited to,cleavable moieties within the linker, such as, for example, a disulfidebond (—S—S—) or an azo bond (—N═N—), which can be cleaved using areducing agent or photolysis. Non-limiting examples of a selectivelycleavable bond include an amido bond can be cleaved for example by theuse of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,and/or photolysis, as well as an ester bond can be cleaved for exampleby acidic or basic hydrolysis.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides.

Mucus: As used herein, “mucus” refers to a natural substance that isviscous and comprises mucin glycoproteins.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Pharmacologic effect: As used herein, a “pharmacologic effect” is ameasurable biologic phenomenon in an organism or system which occursafter the organism or system has been contacted with or exposed to anexogenous agent. Pharmacologic effects may result in therapeuticallyeffective outcomes such as the treatment, improvement of one or moresymptoms, diagnosis, prevention, and delay of onset of disease,disorder, condition or infection. Measurement of such biologic phenomenamay be quantitative, qualitative or relative to another biologicphenomenon. Quantitative measurements may be statistically significant.Qualitative measurements may be by degree or kind and may be at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more different. They maybe observable as present or absent, better or worse, greater or less.Exogenous agents, when referring to pharmacologic effects are thoseagents which are, in whole or in part, foreign to the organism orsystem. For example, modifications to a wild type biomolecule, whetherstructural or chemical, would produce an exogenous agent. Likewise,incorporation or combination of a wild type molecule into or with acompound, molecule or substance not found naturally in the organism orsystem would also produce an exogenous agent. The modified mRNA of thepresent invention, comprise exogenous agents. Examples of pharmacologiceffects include, but are not limited to, alteration in cell count suchas an increase or decrease in neutrophils, reticulocytes, granulocytes,erythrocytes (red blood cells), megakaryocytes, platelets, monocytes,connective tissue macrophages, epidermal langerhans cells, osteoclasts,dendritic cells, microglial cells, neutrophils, eosinophils, basophils,mast cells, helper T cells, suppressor T cells, cytotoxic T cells,natural killer T cells, B cells, natural killer cells, or reticulocytes.Pharmacologic effects also include alterations in blood chemistry, pH,hemoglobin, hematocrit, changes in levels of enzymes such as, but notlimited to, liver enzymes AST and ALT, changes in lipid profiles,electrolytes, metabolic markers, hormones or other marker or profileknown to those of skill in the art.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methyl-pseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits the inclusion of additional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Modified mRNA Production

Modified mRNAs (mmRNA) according to the invention may be made usingstandard laboratory methods and materials. The open reading frame (ORF)of the gene of interest may be flanked by a 5′ untranslated region (UTR)which may contain a strong Kozak translational initiation signal and/oran alpha-globin 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. The modified mRNAs may be modifiedto reduce the cellular innate immune response. The modifications toreduce the cellular response may include pseudouridine (ψ) and5-methyl-cytidine (5meC or m⁵C). (see, Kariko K et al. Immunity23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson BR et al. NAR (2010); each of which are herein incorporated by referencein their entireties).

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the plasmid DNA, it may be reconstitutedand transformed into chemically competent E. coli.

For the present invention, NEB DH5-alpha Competent E. coli are used.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

-   -   1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for        10 minutes.    -   2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell        mixture. Carefully flick the tube 4-5 times to mix cells and        DNA. Do not vortex.    -   3. Place the mixture on ice for 30 minutes. Do not mix.    -   4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.    -   5. Place on ice for 5 minutes. Do not mix.    -   6. Pipette 950 μl of room temperature SOC into the mixture.    -   7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or        rotate.    -   8. Warm selection plates to 37° C.    -   9. Mix the cells thoroughly by flicking the tube and inverting.

Spread 50-100 μl of each dilution onto a selection plate and incubateovernight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmid(an Example of which is shown in FIG. 2) is first linearized using arestriction enzyme such as XbaI. A typical restriction digest with XbaIwill comprise the following: Plasmid 1.0 μg; 10× Buffer 1.0 μl; XbaI 1.5μl; dH₂O up to 10 μl; incubated at 37° C. for 1 hr. If performing at labscale (<5 μg), the reaction is cleaned up using Invitrogen's PURELINK™PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions. Largerscale purifications may need to be done with a product that has a largerload capacity such as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad,Calif.). Following the cleanup, the linearized vector is quantifiedusing the NanoDrop and analyzed to confirm linearization using agarosegel electrophoresis.

The methods described herein to make modified mRNA may be used toproduce molecules of all sizes including long molecules. Modified mRNAusing the described methods has been made for different sized moleculesincluding glucosidase, alpha; acid (GAA) (3.2 kb), cystic fibrosistransmembrane conductance regulator (CFTR) (4.7 kb), Factor VII (7.3kb), lysosomal acid lipase (45.4 kDa), glucocerebrosidase (59.7 kDa) andiduronate 2-sulfatase (76 kDa).

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Sequences used in the steps outlined in Examples 1-5 are shownin Table 4. It should be noted that the start codon (ATG) has beenunderlined in each sequence of Table 4.

TABLE 4 G-CSF Sequences SEQ ID NO Description 3 cDNA sequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA 4cDNA having T7 polymerase site, AfeI and Xba restriction site:TAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA5 Optimized sequence; containing T7 polymerase site,AfeI and Xba restriction site TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA6 mRNA sequence (transcribed)GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAC CAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGC AGCCGUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGA AG

Example 2 PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3 In vitro Transcription

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1. Template cDNA 1.0 μg 2. 10x transcription buffer (400 mM Tris-HCl 2.0μl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3. Custom NTPs (25mM each) 7.2 μl 4. RNase Inhibitor 20 U 5. T7 RNA polymerase 3000 U 6.dH₂0 Up to 20.0 μl. and 7. Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4 Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400 U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂) (12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6 Natural 5′ Caps and 5′ Cap Analogues

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3″-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7 Capping

A. Protein Expression Assay

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 6with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA (3′ O-Me-m7G(5′)ppp(5′)G) cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat equal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 6with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA cap analog or the Cap1 structure crudesynthesis products can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs witha single, consolidated band by electrophoresis correspond to the higherpurity product compared to a synthetic mRNA with multiple bands orstreaking bands. Synthetic mRNAs with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure mRNA population.

C. Cytokine Analysis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 6with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can betransfected into human primary keratinocytes at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Synthetic mRNAs that secrete higherlevels of pro-inflammatory cytokines into the medium would correspond toa synthetic mRNA containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 6with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can beanalyzed for capping reaction efficiency by LC-MS after capped mRNAnuclease treatment. Nuclease treatment of capped mRNAs would yield amixture of free nucleotides and the capped 5′-5-triphosphate capstructure detectable by LC-MS. The amount of capped product on the LC-MSspectra can be expressed as a percent of total mRNA from the reactionand would correspond to capping reaction efficiency. The cap structurewith higher capping reaction efficiency would have a higher amount ofcapped product by LC-MS.

Example 8 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual modified RNAs (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9 Formulation of Modified mRNA Using Lipidoids

Modified mRNAs (mmRNA) are formulated for in vitro experiments by mixingthe mmRNA with the lipidoid at a set ratio prior to addition to cells.In vivo formulation may require the addition of extra ingredients tofacilitate circulation throughout the body. To test the ability of theselipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations was used as astarting point. Initial mmRNA-lipidoid formulations may consist ofparticles composed of 42% lipidoid, 48% cholesterol and 10% PEG, withfurther optimization of ratios possible. After formation of theparticle, mmRNA is added and allowed to integrate with the complex. Theencapsulation efficiency is determined using a standard dye exclusionassays.

Materials and Methods for Examples 10-14

A. Lipid Synthesis

Six lipids, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 andDLin-MC3-DMA, were synthesized by methods outlined in the art in orderto be formulated with modified RNA. DLin-DMA and precursorsweresynthesized as described in Heyes et. al, J. Control Release, 2005, 107,276-287. DLin-K-DMA and DLin-KC2-DMA and precursors were synthesized asdescribed in Semple et. al, Nature Biotechnology, 2010, 28, 172-176.98N12-5 and precursor were synthesized as described in Akinc et. al,Nature Biotechnology, 2008, 26, 561-569.

C12-200 and precursors were synthesized according to the method outlinedin Love et. al, PNAS, 2010, 107, 1864-1869. 2-epoxydodecane (5.10 g,27.7 mmol, 8.2 eq) was added to a vial containing Amine 200 (0.723 g,3.36 mmol, 1 eq) and a stirring bar. The vial was sealed and warmed to80° C. The reaction was stirred for 4 days at 80° C. Then the mixturewas purified by silica gel chromatography using a gradient from puredichloromethane (DCM) to DCM:MeOH 98:2. The target compound was furtherpurified by RP-HPLC to afford the desired compound.

DLin-MC3-DMA and precursors were synthesized according to proceduresdescribed in WO 2010054401 herein incorporated by reference in itsentirety. A mixture of dilinoleyl methanol (1.5 g, 2.8 mmol, 1 eq),N,N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1 eq), DIPEA (0.73 mL,4.2 mmol, 1.5 eq) and TBTU(1.35 g, 4.2 mmol, 1.5 eq) in 10 mL of DMF wasstirred for 10 h at room temperature. Then the reaction mixture wasdiluted in ether and washed with water. The organic layer was dried overanhydrous sodium sulfate, filtrated and concentrated under reducedpressure. The crude product was purified by silica gel chromatographyusing a gradient DCM to DCM:MeOH 98:2. Subsequently the target compoundwas subjected to an additional RP-HPLC purification which was done usinga YMC—Pack C4 column to afford the target compound.

B. Formulation of Modified RNA Nanoparticles

Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine(DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol(Sigma-Aldrich, Taufkirchen, Germany), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG-c-DOMG) (NOF, Bouwelven, Belgium) were prepared at concentrationsof 50 mM in ethanol and stored at −20° C. The lipids were combined toyield molar ratio of 50:10:38.5:1.5 (Lipid:DSPC:Cholesterol:PEG-c-DOMG)and diluted with ethanol to a final lipid concentration of 25 mM.Solutions of modified mRNA at a concentration of 1-2 mg/mL in water werediluted in 50 mM sodium citrate buffer at a pH of 3 to form a stockmodified mRNA solution. Formulations of the lipid and modified mRNA wereprepared by combining the synthesized lipid solution with the modifiedmRNA solution at total lipid to modified mRNA weight ratio of 10:1,15:1, 20:1 and 30:1. The lipid ethanolic solution was rapidly injectedinto aqueous modified mRNA solution to afford a suspension containing33% ethanol. The solutions were injected either manually (MI) or by theaid of a syringe pump (SP) (Harvard Pump 33 Dual Syringe Pump HarvardApparatus Holliston, Mass.).

To remove the ethanol and to achieve the buffer exchange, theformulations were dialyzed twice against phosphate buffered saline(PBS), pH 7.4 at volumes 200-times of the primary product using aSlide-A-Lyzer cassettes (Thermo Fisher Scientific Inc. Rockford, Ill.)with a molecular weight cutoff (MWCO) of 10 kD. The first dialysis wascarried at room temperature for 3 hours and then the formulations weredialyzed overnight at 4° C. The resulting nanoparticle suspension wasfiltered through 0.2 μm sterile filter (Sarstedt, Nümbrecht, Germany)into glass vials and sealed with a crimp closure.

C. Characterization of Formulations

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) was used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy was used to determine the concentrationof modified mRNA nanoparticle formulation. 100 μL of the dilutedformulation in 1×PBS was added to 900 μL of a 4:1 (v/v) mixture ofmethanol and chloroform. After mixing, the absorbance spectrum of thesolution was recorded between 230 nm and 330 nm on a DU 800spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The modified RNA concentration in the nanoparticle formulationwas calculated based on the extinction coefficient of the modified RNAused in the formulation and on the difference between the absorbance ata wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.)was used to evaluate the encapsulation of modified RNA by thenanoparticle. The samples were diluted to a concentration ofapproximately 5 μg/mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).50 μL of the diluted samples were transferred to a polystyrene 96 wellplate, then either 50 μL of TE buffer or 50 μL of a 2% Triton X-100solution was added. The plate was incubated at a temperature of 37° C.for 15 minutes. The RIBOGREEN® reagent was diluted 1:100 in TE buffer,100 μL of this solution was added to each well. The fluorescenceintensity was measured using a fluorescence plate reader (Wallac Victor1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at an excitationwavelength of ˜480 nm and an emission wavelength of ˜520 nm. Thefluorescence values of the reagent blank were subtracted from that ofeach of the samples and the percentage of free modified RNA wasdetermined by dividing the fluorescence intensity of the intact sample(without addition of Triton X-100) by the fluorescence value of thedisrupted sample (caused by the addition of Triton X-100).

D. In Vitro Incubation

Human embryonic kidney epithelial (HEK293) and hepatocellular carcinomaepithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) wereseeded on 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany)and plates for HEK293 cells were precoated with collagen type1. HEK293were seeded at a density of 30,000 and HepG2 were seeded at a density of35,000 cells per well in 100 μl cell culture medium. For HEK293 the cellculture medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mMSodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin,Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich, Munich,Germany) and for HepG2 the culture medium was MEM (Gibco LifeTechnologies, Darmstadt, Germany), 10% FCS adding 2 mM L-Glutamine, 1 mMSodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin,Germany. Formulations containing mCherry mRNA (mRNA sequence shown inSEQ ID NO: 7; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1); were added in quadruplicates directly afterseeding the cells and incubated. The mCherry cDNA with the T7 promoter,5′ untranslated region (UTR) and 3′ UTR used in in vitro transcription(IVT) is given in SEQ ID NO: 8. The mCherry mRNA was modified with 5meCat each cytosine and pseudouridine replacement at each uridine site.

Cells were harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells were trypsinized with ½ volume Trypsin/EDTA (BiochromAG, Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted toa flow cytometer measurement with a 532 nm excitation laser and the610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton DickinsonGmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of allevents and the standard deviation of four independent wells arepresented in for samples analyzed.

Example 10 Purification of Nanoparticle Formulations

Nanoparticle formulations of DLin-KC2-DMA and 98N12-5 in HEK293 andHepG2 were tested to determine if the mean fluorescent intensity (MFI)was dependent on the lipid to modified RNA ratio and/or purification.Three formulations of DLin-KC2-DMA and two formulations of 98N12-5 wereproduced using a syringe pump to the specifications described in Table5. Purified samples were purified by SEPHADEX™ G-25 DNA grade (GEHealthcare, Sweden). Each formulation before and after purification (aP)was tested at concentration of 250 ng modified RNA per well in a 24 wellplate. The percentage of cells that are positive for the marker for FL4channel (% FL4-positive) when analyzed by the flow cytometer for eachformulation and the background sample and the MFI of the marker for theFL4 channel for each formulation and the background sample are shown inTable 6. The formulations which had been purified had a slightly higherMFI than those formulations tested before purification.

TABLE 5 Formulations Formulation # Lipid Lipid/RNA wt/wt Mean size (nm)NPA-001-1 DLin-KC2-DMA 10 155 nm PDI: 0.08 NPA-001-1 aP DLin-KC2-DMA 10141 nm PDI: 0.14 NPA-002-1 DLin-KC2-DMA 15 140 nm PDI: 0.11 NPA-002-1 aPDLin-KC2-DMA 15 125 nm PDI: 0.12 NPA-003-1 DLin-KC2-DMA 20 114 nm PDI:0.08 NPA-003-1 aP DLin-KC2-DMA 20 104 nm PDI: 0.06 NPA-005-1 98N12-5 15127 nm PDI: 0.12 NPA-005-1 aP 98N12-5 15 134 nm PDI: 0.17 NPA-006-198N12 20 126 nm PDI: 0.08 NPA-006-1 aP 98N12 20 118 nm PDI: 0.13

TABLE 6 HEK293 and HepG2, 24-well, 250 ng Modified RNA/well %FL4-positive FL4 MFI Formulation HEK293 HepG2 HEK293 HepG2 Untreated0.33 0.40 0.25 0.30 NPA-001-1 62.42 5.68 1.49 0.41 NPA-001-ap 87.32 9.023.23 0.53 NPA-002-1 91.28 9.90 4.43 0.59 NPA-002-ap 92.68 14.02 5.070.90 NPA-003-1 87.70 11.76 6.83 0.88 NPA-003-ap 88.88 15.46 8.73 1.06NPA-005-1 50.60 4.75 1.83 0.46 NPA-005-ap 38.64 5.16 1.32 0.46 NPA-006-154.19 13.16 1.30 0.60 NPA-006-ap 49.97 13.74 1.27 0.61

Example 11 Concentration Response Curve

Nanoparticle formulations of 98N12-5 (NPA-005) and DLin-KC2-DMA(NPA-003) were tested at varying concentrations to determine the MFI ofFL4 or mCherry (mRNA sequence shown in SEQ ID NO: 7; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) over a range of doses.The formulations tested are outlined in Table 7. To determine theoptimal concentration of nanoparticle formulations of 98N12-5, varyingconcentrations of formulated modified RNA (100 ng, 10 ng, 1.0 ng, 0.1 ngand 0.01 ng per well) were tested in a 24-well plate of HEK293, and theresults of the FL4 MFI of each dose are shown in Table 8. Likewise, todetermine the optimal concentration of nanoparticle formulations ofDLin-KC2-DMA, varying concentrations of formulated modified RNA (250 ng100 ng, 10 ng, 1.0 ng, 0.1 ng and 0.01 ng per well) were tested in a24-well plate of HEK293, and the results of the FL4 MFI of each dose areshown in Table 9. Nanoparticle formulations of DLin-KC2-DMA were alsotested at varying concentrations of formulated modified RNA (250 ng, 100ng and 30 ng per well) in a 24 well plate of HEK293, and the results ofthe FL4 MFI of each dose are shown in Table 10. A dose of 1 ng/well for98N12-5 and a dose of 10 ng/well for DLin-KC2-DMA were found to resemblethe FL4 MFI of the background.

To determine how close the concentrations resembled the background, weutilized a flow cytometer with optimized filter sets for detection ofmCherry expression, and were able to obtain results with increasedsensitivity relative to background levels. Doses of 25 ng/well, 0.25ng/well, 0.025 ng/well and 0.0025 ng/well were analyzed for 98N12-5(NPA-005) and DLin-KC2-DMA (NPA-003) to determine the MFI of mCherry. Asshown in Table 11, the concentration of 0.025 ng/well and lesserconcentrations are similar to the background MFI level of mCherry whichis about 386.125.

TABLE 7 Formulations Formulation # NPA-003 NPA-005 Lipid DLin-KC2-DMA98N12-5 Lipid/RNA 20 15 wt/wt Mean size 114 nm 106 nm PDI: 0.08 PDI:0.12

TABLE 8 HEK293, NPA-005, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.246 NPA-005 100 ng 2.2175 NPA-005 10 ng 0.651 NPA-005 1.0 ng0.28425 NPA-005 0.1 ng 0.27675 NPA-005 0.01 ng 0.2865

TABLE 9 HEK293, NPA-003, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.3225 NPA-003 250 ng 2.9575 NPA-003 100 ng 1.255 NPA-003 10 ng0.40025 NPA-003 1 ng 0.33025 NPA-003 0.1 ng 0.34625 NPA-003 0.01 ng0.3475

TABLE 10 HEK293, NPA-003, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.27425 NPA-003 250 ng 5.6075 NPA-003 100 ng 3.7825 NPA-003 30ng 1.5525

TABLE 11 Concentration and MFI MFI mCherry Formulation NPA-003 NPA-005   25 ng/well 11963.25 12256.75  0.25 ng/well 1349.75 2572.75  0.025ng/well 459.50 534.75 0.0025 ng/well 310.75 471.75

Example 12 Manual Injection and Syringe Pump Formulations

Two formulations of DLin-KC2-DMA and 98N12-5 were prepared by manualinjection (MI) and syringe pump injection (SP) and analyzed along with abackground sample to compare the MFI of mCherry (mRNA sequence shown inSEQ ID NO: 7; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) of the different formulations. Table 12 shows that thesyringe pump formulations had a higher MFI as compared to the manualinjection formulations of the same lipid and lipid/RNA ratio.

TABLE 12 Formulations and MFI Lipid/ Method of Formulation RNA Mean sizeformula- # Lipid wt/wt (nm) tion MFI Untreated N/A N/A N/A N/A 674.67Control NPA-002 DLin-KC2- 15 140 nm MI 10318.25 DMA PDI: 0.11 NPA-002-2DLin-KC2- 15 105 nm SP 37054.75 DMA PDI: 0.04 NPA-003 DLin-KC2- 20 114nm MI 22037.5 DMA PDI: 0.08 NPA-003-2 DLin-KC2- 20  95 nm SP 37868.75DMA PDI: 0.02 NPA-005 98N12-5 15 127 nm MI 11504.75 PDI: 0.12 NPA-005-298N12-5 15 106 nm SP 9343.75 PDI: 0.07 NPA-006 98N12-5 20 126 nm MI11182.25 PDI: 0.08 NPA-006-2 98N12-5 20  93 nm SP 5167 PDI: 0.08

Example 13 LNP Formulations

Formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 andDLin-MC3-DMA were incubated at a concentration of 60 ng/well or 62.5ng/well in a plate of HEK293 and 62.5 ng/well in a plate of HepG2 cellsfor 24 hours to determine the MFI of mCherry (mRNA sequence shown in SEQID NO: 7; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) for each formulation. The formulations tested areoutlined in Table 13 below. As shown in Table 14 for the 60 ng/well andTables 15, 16, 17 and 18 for the 62.5 ng/well, the formulation ofNPA-003 and NPA-018 have the highest mCherry MFI and the formulations ofNPA-008, NPA-010 and NPA-013 are most the similar to the backgroundsample mCherry MFI value.

TABLE 13 Formulations Formulation # Lipid Lipid/RNA wt/wt Mean size (nm)NPA-001 DLin-KC2-DMA 10 155 nm PDI: 0.08 NPA-002 DLin-KC2-DMA 15 140 nmPDI: 0.11 NPA-002-2 DLin-KC2-DMA 15 105 nm PDI: 0.04 NPA-003DLin-KC2-DMA 20 114 nm PDI: 0.08 NPA-003-2 DLin-KC2-DMA 20  95 nm PDI:0.02 NPA-005 98N12-5 15 127 nm PDI: 0.12 NPA-006 98N12-5 20 126 nm PDI:0.08 NPA-007 DLin-DMA 15 148 nm PDI: 0.09 NPA-008 DLin-K-DMA 15 121 nmPDI: 0.08 NPA-009 C12-200 15 138 nm PDI: 0.15 NPA-010 DLin-MC3-DMA 15126 nm PDI: 0.09 NPA-012 DLin-DMA 20  86 nm PDI: 0.08 NPA-013 DLin-K-DMA20 104 nm PDI: 0.03 NPA-014 C12-200 20 101 nm PDI: 0.06 NPA-015DLin-MC3-DMA 20 109 nm PDI: 0.07

TABLE 14 HEK293, 96-well, 60 ng Modified RNA/well Formulation MFImCherry Untreated 871.81 NPA-001 6407.25 NPA-002 14995 NPA-003 29499.5NPA-005 3762 NPA-006 2676 NPA-007 9905.5 NPA-008 1648.75 NPA-009 2348.25NPA-010 4426.75 NPA-012 11466 NPA-013 2098.25 NPA-014 3194.25 NPA-01514524

TABLE 15 HEK293, 62.5 ng/well Formulation MFI mCherry Untreated 871.81NPA-001 6407.25 NPA-002 14995 NPA-003 29499.5 NPA-005 3762 NPA-006 2676NPA-007 9905.5 NPA-008 1648.75 NPA-009 2348.25 NPA-010 4426.75 NPA-01211466 NPA-013 2098.25 NPA-014 3194.25 NPA-015 14524

TABLE 16 HEK293, 62.5 ng/well Formulation MFI mCherry Untreated 295NPA-007 3504 NPA-012 8286 NPA-017 6128 NPA-003-2 17528 NPA-018 34142NPA-010 1095 NPA-015 5859 NPA-019 3229

TABLE 17 HepG2, 62.5 ng/well Formulation MFI mCherry Untreated 649.94NPA-001 6006.25 NPA-002 8705 NPA-002-2 15860.25 NPA-003 15059.25NPA-003-2 28881 NPA-005 1676 NPA-006 1473 NPA-007 15678 NPA-008 2976.25NPA-009 961.75 NPA-010 3301.75 NPA-012 18333.25 NPA-013 5853 NPA-0142257 NPA-015 16225.75

TABLE 18 HepG2, 62.5 ng/well Formulation MFI mCherry Untreated control656 NPA-007 16798 NPA-012 21993 NPA-017 20377 NPA-003-2 35651 NPA-01840154 NPA-010 2496 NPA-015 19741 NPA-019 16373

Example 14 In Vivo Formulation Studies

Rodents (n=5) are administered intravenously, subcutaneously orintramuscularly a single dose of a formulation containing at least onemodified mRNA and a lipid. The modified mRNA administered to the rodentsis selected from G-CSF (mRNA sequence shown in SEQ ID NO: 6; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),erythropoietin (EPO) (mRNA sequence shown in SEQ ID NO: 9; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),Factor IX (mRNA shown in SEQ ID NO: 10; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) or mCherry (mRNAsequence shown in SEQ ID NO: 7; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1). The erythropoietincDNA with the T7 promoter, 5′ untranslated region (UTR) and 3′ UTR usedin in vitro transcription (IVT) is given in SEQ ID NO: 11 and SEQ ID NO:12.

Each formulation also contains a lipid which is selected from one ofDLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA,reLNP, ATUPLEX®, DACC, and DBTC. The rodents are injected with 100 ug,10 ug or 1 ug of the formulated modified mRNA and samples are collectedat specified time intervals.

Serum from the rodents administered formulations containing human G-CSFmodified mRNA are measured by specific G-CSF ELISA and serum from miceadministered human factor IX modified RNA is analyzed by specific factorIX ELISA or chromogenic assay. The liver and spleen from the miceadministered with mCherry modified mRNA are analyzed byimmunohistochemistry (IHC) or fluorescence-activated cell sorting(FACS). As a control, a group of mice are not injected with anyformulation and their serum and tissue are collected analyzed by ELISA,FACS and/or IHC.

A. Time Course

The rodents are administered formulations containing at least onemodified mRNA to study the time course of protein expression for theadministered formulation. The rodents are bled at specified timeintervals prior to and after administration of the modified mRNAformulations to determine protein expression and complete blood count.Samples are also collected from the site of administration of rodentsadministered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

B. Dose Response

The rodents are administered formulations containing at least onemodified mRNA to determine dose response of each formulation. Therodents are bled at specified time intervals prior to and afteradministration of the modified mRNA formulations to determine proteinexpression and complete blood count. The rodents are also sacrified toanalyze the effect of the modified mRNA formulation on the internaltissue. Samples are also collected from the site of administration ofrodents administered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

C. Toxicity

The rodents are administered formulations containing at least onemodified mRNA to study toxicity of each formulation. The rodents arebled at specified time intervals prior to and after administration ofthe modified mRNA formulations to determine protein expression andcomplete blood count. The rodents are also sacrificed to analyze theeffect of the modified mRNA formulation on the internal tissue. Samplesare also collected from the site of administration of rodentsadministered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

Example 15 PLGA Microsphere Formulations

Optimization of parameters used in the formulation of PLGA microspheresmay allow for tunable release rates and high encapsulation efficiencieswhile maintaining the integrity of the modified RNA encapsulated in themicrospheres. Parameters such as, but not limited to, particle size,recovery rates and encapsulation efficiency may be optimized to achievethe optimal formulation.

A. Synthesis of PLGA Microspheres

Polylacticglycolic acid (PLGA) microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23 k)dichloromethane and water. Briefly, 0.1 ml of water (W1) was added to 2ml of PLGA dissolved in dichloromethane (DCM) (O1) at concentrationsranging from 50-200 mg/ml of PLGA. The W1/O1 emulsion was homogenized(IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed 4 (˜15,000rpm). The W1/O1 emulsion was then added to 100 to 200 ml of 0.3 to 1%PVA (W2) and homogenized for 1 minute at varied speeds. Formulationswere left to stir for 3 hours and then washed by centrifugation (20-25min, 4,000 rpm, 4° C.). The supernatant was discarded and the PLGApellets were resuspended in 5-10 ml of water, which was repeated 2×.Average particle size (represents 20-30 particles) for each formulationwas determined by microscopy after washing. Table 19 shows that anincrease in the PLGA concentration led to larger sized microspheres. APLGA concentration of 200 mg/mL gave an average particle size of 14.8μm, 100 mg/mL was 8.7 μm, and 50 mg/mL of PLGA gave an average particlesize of 4.0 μm.

TABLE 19 Varied PLGA Concentration PLGA PVA O1 Concen- W2 Con- AverageSample Volume tration Volume centration Size ID (mL) (mg/mL) (mL) (%)Speed (μm) 1 2 200 100 0.3 5 14.8 2 2 100 100 0.3 5 8.7 3 2 50 100 0.3 54.0

Table 20 shows that decreasing the homogenization speed from 5 (˜20,000rpm) to speed 4 (˜15,000 rpm) led to an increase in particle size from14.8 μm to 29.7 μm.

TABLE 20 Varied Homogenization Speed PLGA PVA O1 Con- W2 Con- AverageSample Volume centration Volume centration Size ID (mL) (mg/mL) (mL) (%)Speed (μm) 1 2 200 100 0.3 5 14.8 4 2 200 100 0.3 4 29.7

Table 21 shows that increasing the W2 volume (i.e. increasing the ratioof W2:O1 from 50:1 to 100:1), decreased average particle size slightly.Altering the PVA concentration from 0.3 to 1 wt % had little impact onPLGA microsphere size.

TABLE 21 Varied W2 Volume and Concentration PLGA PVA O1 Con- W2 Con-Average Sample Volume centration Volume centration Size ID (mL) (mg/mL)(mL) (%) Speed (μm) 1 2 200 100 0.3 5 14.8 5 2 200 200 0.3 5 11.7 6 2200 190 0.3 5 11.4 7 2 200 190 1.0 5 12.3

B. Encapsulation of Modified mRNA

Modified G-CSF mRNA (SEQ ID NO: 6; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) was dissolved in water at aconcentration of 2 mg/ml (W3). Three batches of PLGA microsphereformulations were made as described above with the following parameters:0.1 ml of W3 at 2 mg/ml, 1.6 ml of O1 at 200 mg/ml, 160 ml of W2 at 1%,and homogenized at a speed of 4 for the first emulsion (W3/O1) andhomogenized at a speed of 5 for the second emulstion (W3/O1/W2). Afterwashing by centrifugation, the formulations were frozen in liquidnitrogen and then lyophilized for 3 days. To test the encapsulationefficiency of the formulations, the lyophilized material wasdeformulated in DCM for 6 hours followed by an overnight extraction inwater. The modified RNA concentration in the samples was then determinedby OD260. Encapsulation efficiency was calculated by taking the actualamount of modified RNA and dividing by the starting amount of modifiedRNA. In the three batches tested, there was an encapsulation efficiencyof 59.2, 49.8 and 61.3.

C. Integrity of Modified mRNA Encapsulated in PLGA Microspheres

Modified Factor IX mRNA (SEQ ID NO: 10; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) was dissolved in water at variedconcentrations (W4) to vary the weight percent loading in theformulation (mg modified RNA/mg PLGA*100) and to determine encapsulationefficiency. The parameters in Table 22 were used to make four differentbatches of PLGA microsphere formulations with a homogenization speed of4 for the first emulstion (W4/O1) and a homogenization speed of 5 forthe second emulsion (W4/O1/W2).

TABLE 22 Factor IX PLGA Microsphere Formulation Parameters Factor W4Factor IX IX O1 PLGA W2 PVA Weight % Volume Concentration Amount VolumeConcentration Volume Concentration (wt %) ID (uL) (mg/ml) (ug) (ml)(mg/ml) (ml) (%) Loading A 100 2.0 200.0 2.0 200 200 1.0 0.05 B 100 4.0400.0 2.0 200 200 1.0 0.10 C 400 2.0 800.0 2.0 200 200 1.0 0.20 D 4004.0 1600.0 2.0 200 200 1.0 0.40

After lyophilization, PLGA microspheres were weighed out in 2 mleppendorf tubes to correspond to ˜10 ug of modified RNA. Lyophilizationwas found to not destroy the overall structure of the PLGA microspheres.To increase weight percent loading (wt %) for the PLGA microspheres,increasing amounts of modified RNA were added to the samples. PLGAmicrospheres were deformulated by adding 1.0 ml of DCM to each tube andthen shaking the samples for 6 hours. For modified RNA extraction, 0.5ml of water was added to each sample and the samples were shakenovernight before the concentration of modified RNA in the samples wasdetermined by OD260. To determine the recovery of the extractionprocess, unformulated Factor IX modified RNA (SEQ ID NO: 10; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) (deformulationcontrol) was spiked into DCM and was subjected to the deformulationprocess. Table 23 shows the loading and encapsulation efficiency for thesamples. All encapsulation efficiency samples were normalized to thedeformulation control.

TABLE 23 Weight Percent Loading and Encapsulation Efficiency TheoreticalActual modified modified RNA RNA Encapsulation ID loading (wt %) loading(wt %) Efficiency (%) A 0.05 0.06 97.1 B 0.10 0.10 85.7 C 0.20 0.18 77.6D 0.40 0.31 68.1 Control — — 100.0

D. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified RNA (SEQ ID NO: 10)were deformulated as described above and the integrity of the extractedmodified RNA was determined by automated electrophoresis (Bio-RadExperion). The extracted modified mRNA was compared against unformulatedmodified mRNA and the deformulation control in order to test theintegrity of the encapsulated modified mRNA. As shown in FIG. 3, themajority of modRNA was intact for batch ID A, B, C and D, for thedeformulated control (Deform control) and the unformulated control(Unform control).

E. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified RNA (SEQ ID NO: 10;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) weredeformulated as described above and the protein expression of theextracted modified RNA was determined by an in vitro transfection assay.HEK293 cells were reverse transfected with 250 ng of Factor IX modifiedRNA complexed with RNAiMAX (Invitrogen) in triplicate.

Factor IX modified RNA was diluted in nuclease-free water to aconcentration of 25 ng/μl and RNAiMAX was diluted 13.3× in serum-freeEMEM. Equal volumes of diluted modified RNA and diluted RNAiMAX weremixed together and were allowed to stand for 20 to 30 minutes at roomtemperature. Subsequently, 20 μl of the transfection mix containing 250ng of Factor IX modified RNA was added to 80 μl of a cell suspensioncontaining 30,000 cells. Cells were then incubated for 16 h in ahumidified 37° C./5% CO2 cell culture incubator before harvesting thecell culture supernatant. Factor IX protein expression in the cellsupernatant was analyzed by an ELISA kit specific for Factor IX(Molecular Innovations, Cat # HFIXKT-TOT) and the protein expression isshown in Table 24. In all PLGA microsphere batches tested, Factor IXmodified RNA remained active and expressed Factor IX protein afterformulation in PLGA microspheres and subsequent deformulation.

TABLE 24 Protein Expression Factor IX Protein Sample Expression (ng/ml)Batch A 0.83 Batch B 1.83 Batch C 1.54 Batch D 2.52 Deformulated Control4.34 Unformulated Control 3.35

F. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA micropsheres formulated with Factor IX modified RNA (SEQ ID NO: 10;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) wereresuspended in water to a PLGA microsphere concentration of 24 mg/ml.After resuspension, 150 ul of the PLGA microsphere suspension wasaliquoted into eppendorf tubes. Samples were kept incubating and shakingat 37° C. during the course of the study. Triplicate samples were pulledat 0.2, 1, 2, 8, 14, and 21 days. To determine the amount of modifiedRNA released from the PLGA microspheres, samples were centrifuged, thesupernatant was removed, and the modified RNA concentration in thesupernatant was determined by OD 260. The percent release, shown inTable 25, was calculated based on the total amount of modified RNA ineach sample. After 31 days, 96% of the Factor IX modified RNA wasreleased from the PLGA microsphere formulations.

TABLE 25 Percent Release Time (days) % Release 0 0.0 0.2 27.0 1 37.7 245.3 4 50.9 8 57.0 14 61.8 21 75.5 31 96.4

G. Particle Size Reproducibility of PLGA Microspheres

Three batches of Factor IX modified RNA (SEQ ID NO: 10 polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) PLGA microspheres weremade using the same conditions described for Batch D, shown in Table 22,(0.4 ml of W4 at 4 mg/ml, 2.0 ml of O1 at 200 mg/ml, 200 ml of W2 at 1%,and homogenized at a speed of 5 for the W4/O1/W2 emulsion). To improvethe homogeneity of the PLGA microsphere suspension, filtration wasincorporated prior to centrifugation. After stirring for 3 hours andbefore centrifuging, all formulated material was passed through a 100 μmnylon mesh strainer (Fisherbrand Cell Strainer, Cat #22-363-549) toremove larger aggregates. After washing and resuspension with water,100-200 μl of a PLGA microspheres sample was used to measure particlesize of the formulations by laser diffraction (Malvern Mastersizer2000).The particle size of the samples is shown in Table 26.

TABLE 26 Particle Size Summary Volume Weighted ID D10 (μm) D50 (μm) D90(μm) Mean (um) Filtration Control 19.2 62.5 722.4 223.1 No A 9.8 31.665.5 35.2 Yes B 10.5 32.3 66.9 36.1 Yes C 10.8 35.7 79.8 41.4 Yes

Results of the 3 PLGA microsphere batches using filtration were comparedto a PLGA microsphere batch made under the same conditions withoutfiltration. The inclusion of a filtration step before washing reducedthe mean particle size and demonstrated a consistent particle sizedistribution between 3 PLGA microsphere batches.

H. Serum Stability of Factor IX PLGA Microspheres

Factor IX mRNA RNA (SEQ ID NO: 10 polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) in buffer (TE) or 90% serum (Se), orFactor IX mRNA in PLGA in buffer, 90% serum or 1% serum was incubated inbuffer, 90% serum or 1% serum at an mRNA concentration of 50 ng/ul in atotal volume of 70 ul. The samples were removed at 0, 30, 60 or 120minutes. RNases were inactivated with proteinase K digestion for 20minutes at 55° C. by adding 25 ul of 4× proteinase K buffer (0.4 ml 1MTRIS-HCl pH 7.5, 0.1 ml 0.5M EDTA, 0.12 ml 5M NaCl, and 0.4 ml 10% SDS)and 8 ul of proteinase K at 20 mg/ml. The Factor IX mRNA wasprecipitated (add 250 ul 95% ethanol for 1 hour, centrifuge for 10 minat 13 k rpm and remove supernatant, add 200 ul 70% ethanol to thepellet, centrifuge again for 5 min at 13 k rpm and remove supernatantand resuspend the pellet in 70 ul water) or extracted from PLGAmicrospheres (centrifuge 5 min at 13 k rpm and remove supernatant, washpellet with 1 ml water, centrifuge 5 min at 13 k rpm and removesupernatant, add 280 ul dichloromethane to the pellet and shake for 15minutes, add 70 ul water and then shake for 2 hours and remove theaqueous phase) before being analyzed by bioanalyzer. PLGA microspheresprotect Factor IX modified mRNA from degradation in 90% and 1% serumover 2 hours. Factor IX modified mRNA completely degrades in 90% serumat the initial time point.

Example 16 Lipid Nanoparticle In Vivo Studies

G-CSF (cDNA with the T7 promoter, 5′ Untranslated region (UTR) and 3′UTRused in in vitro transcription is given in SEQ ID NO: 5. mRNA sequenceshown in SEQ ID NO: 6; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap 1; fully modified with 5-methylcytosineand pseudouridine) and Factor IX (cDNA with the T7 promoter, 5′ UTR and3′UTR used in in vitro transcription is given in SEQ ID NO: 13. mRNAsequence shown in SEQ ID NO:10; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap 1; fully modified with5-methylcytosine and pseudouridine) modified mRNA were formulated aslipid nanoparticles (LNPs) using the syringe pump method. The LNPs wereformulated at a 20:1 weight ratio of total lipid to modified mRNA with afinal lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). Formulations, listed inTable 27, were characterized by particle size, zeta potential, andencapsulation.

TABLE 27 Formulations Formulation # NPA-029-1 NPA-030-1 Modified mRNAFactor IX G-CSF Mean size 91 nm 106 nm PDI: 0.04 PDI: 0.06 Zeta at pH7.4 1.8 mV 0.9 mV Encaps. 92% 100% (RiboGr)

LNP formulations were administered to mice (n=5) intravenously at amodified mRNA dose of 100, 10, or 1 ug. Mice were sacrificed at 8 hrsafter dosing. Serum was collected by cardiac puncture from mice thatwere administered with G-CSF or Factor IX modified mRNA formulations.Protein expression was determined by ELISA.

There was no significant body weight loss (<5%) in the G-CSF or FactorIX dose groups. Protein expression for G-CSF or Factor IX dose groupswas determined by ELISA from a standard curve. Serum samples werediluted (about 20-2500× for G-CSF and about 10-250× for Factor IX) toensure samples were within the linear range of the standard curve. Asshown in Table 28, G-CSF protein expression determined by ELISA wasapproximately 17, 1200, and 4700 ng/ml for the 1, 10, and 100 ug dosegroups, respectively. As shown in Table 29, Factor IX protein expressiondetermined by ELISA was approximately 36, 380, and 3000-11000 ng/ml forthe 1, 10, and 100 ug dose groups, respectively.

TABLE 28 G-CSF Protein Expression Sample Dose (ug) Conc (ng/ml) DilutionFactor Volume 1 17.73  20x   5 ul 10 1204.82 2500x 0.04 ul 100 4722.202500x 0.04 ul

TABLE 29 Factor IX Protein Expression Sample Dose (ug) Conc (ng/ml)Dilution Factor Volume  1 36.05 10x 5 ul  10 383.04 10x 5 ul  100*3247.75 50x 1 ul  100* 11177.20 250x  0.2 ul  

As shown in Table 30, the LNP formulations described above have about a10,000-100,000-fold increase in protein production compared to anadministration of an intravenous (IV)-lipoplex formulation for the samedosage of modified mRNA and intramuscular (IM) or subcutaneous (SC)administration of the same dose of modified mRNA in saline. As used inTable 30, the symbol “˜” means about.

TABLE 30 Protein Production Serum Concentration (pg/ml) G-CSF Dose (ug)8-12 hours after administration IM 100 ~20-80 SC 100 ~10-40 IV(Lipoplex) 100 ~30 IV (LNP) 100 ~5,000,000 IV (LNP) 10 ~1,000,000 IV(LNP) 1 ~20,000 Serum Concentration (ng/ml) Factor IX Dose (ug) 8-12hours after administration IM 2 × 100 ~1.6 ng/ml IV (LNP) 100~3,000-10,000 ng/ml IV (LNP) 10 ~400 ng/ml IV (LNP) 1 ~40 ng/ml

Materials and Methods for Examples 17-22

G-CSF (mRNA sequence shown in SEQ ID NO: 6; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap 1; fully modifiedwith 5-methylcytosine and pseudouridine) and EPO (mRNA sequence shown inSEQ ID NO: 9; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap 1; fully modified with 5-methylcytosine andpseudouridine) modified mRNA were formulated as lipid nanoparticles(LNPs) using the syringe pump method. The LNPs were formulated at a 20:1weight ratio of total lipid to modified mRNA with a final lipid molarratio of 50:10:38.5:1.5 (DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG).Formulations, listed in Table 31, were characterized by particle size,zeta potential, and encapsulation.

TABLE 31 Formulations Formulation # NPA-030-2 NPA-060-1 Modified mRNAG-CSF EPO Mean size 84 nm 85 nm PDI: 0.04 PDI: 0.03 Zeta at pH 7.4 0.8mV 1.5 mV Encapsulation 95% 98% (RiboGreen)

Example 17 Lipid Nanoparticle In Vivo Studies with Modified mRNA

LNP formulations, shown in Table 31 (above), were administered to rats(n=5) intravenously (IV), intramuscularly (IM) or subcutaneously (SC) ata single modified mRNA dose of 0.05 mg/kg. A control group of rats (n=4)was untreated. The rats were bled at 2 hours, 8 hours, 24 hours, 48hours and 96 hours and after they were administered with G-CSF or EPOmodified mRNA formulations to determine protein expression using ELISA.The rats administered EPO modified mRNA intravenously were also bled at7 days.

As shown in Table 32, EPO protein expression in the rats intravenouslyadministered modified EPO mRNA was detectable out to 5 days. G-CSF inthe rats intravenously administered modified G-CSF mRNA was detectableto 7 days. Subcutaneous and intramuscular administration of EPO modifiedmRNA was detectable to at least 24 hours and G-CSF modified mRNA wasdetectable to at least 8 hours. In Table 32, “OSC” refers to values thatwere outside the standard curve and “NT” means not tested.

TABLE 32 G-CSF and EPO Protein Expression EPO Serum G-CSF Serum RouteTime Concentration (pg/ml) Concentration (pg/ml) IV 2 hours 36,981.031,331.9 IV 8 hours 62,053.3 70,532.4 IV 24 hours 42,077.0 5,738.6 IV 48hours 5,561.5 233.8 IV 5 days 0.0 60.4 IV 7 days 0.0 NT IM 2 hours1395.4 1620.4 IM 8 hours 8974.6 7910.4 IM 24 hours 4678.3 893.3 IM 48hours NT OSC IM 5 days NT OSC SC 2 hours 386.2 80.3 SC 8 hours 985.6164.2 SC 24 hours 544.2 OSC SC 48 hours NT OSC SC 5 days NT OSCUntreated All bleeds 0 0

Example 18 Time Course In Vivo Study

LNP formulations, shown in Table 31 (above), were administered to mice(n=5) intravenously (IV) at a single modified mRNA dose of 0.5, 0.05 or0.005 mg/kg. The mice were bled at 8 hours, 24 hours, 72 hours and 6days after they were administered with G-CSF or EPO modified mRNAformulations to determine protein expression using ELISA.

As shown in Table 33, EPO and G-CSF protein expression in the miceadministered with the modified mRNA intravenously was detectable out to72 hours for the mice dosed with 0.005 mg/kg and 0.05 mg/kg of modifiedmRNA and out to 6 days for the mice administered the EPO modified mRNA.In Table 33, “>” means greater than and “ND” means not detected.

TABLE 33 Protein Expression Dose EPO Serum G-CSF Serum (mg/kg) TimeConcentration (pg/ml) Concentration (pg/ml) 0.005 8 hours 12,508.311,550.6 0.005 24 hours 6,803.0 5,068.9 0.005 72 hours ND ND 0.005 6days ND ND 0.05 8 hours 92,139.9 462,312.5 0.05 24 hours 54,389.480,903.8 0.05 72 hours ND ND 0.05 6 days ND ND 0.5 8 hours498,515.3 >1,250,000 0.5 24 hours 160,566.3 495,812.5 0.5 72 hours3,492.5 1,325.6 0.5 6 days 21.2 ND

Example 19 LNP Formulations In Vivo Study in Rodents

A. LNP Formulations in Mice

LNP formulations, shown in Table 31 (above), were administered to mice(n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg or0.005 mg/kg. There was also 3 control groups of mice (n=4) that wereuntreated. The mice were bled at 2 hours, 8 hours, 24 hours, 48 hoursand 72 hours after they were administered with G-CSF or EPO modifiedmRNA formulations to determine the protein expression. Proteinexpression of G-CSF and EPO were determined using ELISA.

As shown in Table 34, EPO and G-CSF protein expression in the mice wasdetectable at least out to 48 hours for the mice that received a dose of0.005 mg/kg modified RNA and 72 hours for the mice that received a doseof 0.05 mg/kg modified RNA. In Table 34, “OSC” refers to values thatwere outside the standard curve and “NT” means not tested.

TABLE 34 Protein Expression in Mice Dose EPO Serum G-CSF Serum (mg/kg)Time Concentration (pg/ml) Concentration (pg/ml) 0.005 2 hours OSC3,447.8 0.005 8 hours 1,632.8 11,454.0 0.005 24 hours 1,141.0 4,960.20.005 48 hours 137.4 686.4 0.005 72 hours 0 NT 0.05 2 hours 10,027.320,951.4 0.05 8 hours 56,547.2 70,012.8 0.05 24 hours 25,027.3 19,356.20.05 48 hours 1,432.3 1,963.0 0.05 72 hours 82.2 47.3

B. LNP Formulations in Rats

LNP formulations, shown in Table 31 (above), are administered to rats(n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg.There is also a control group of rats (n=4) that are untreated. The ratsare bled at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 7 days and14 days after they were administered with G-CSF or EPO modified mRNAformulations to determine the protein expression. Protein expression ofG-CSF and EPO are determined using ELISA.

Example 20 Early Time Course Study of LNPs

LNP formulations, shown in Table 31 (above), are administered to mammalsintravenously (IV), intramuscularly (IM) or subcutaneously (SC) at asingle modified mRNA dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg. Acontrol group of mammals are not treated. The mammals are bled at 5minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5hours and/or 2 hours after they are administered with the modified mRNALNP formulations to determine protein expression using ELISA. Themammals are also bled to determine the complete blood count such as thegranulocyte levels and red blood cell count.

Example 21 Non-Human Primate In Vivo Study

LNP formulations, shown in Table 31 (above), were administered tonon-human primates (NHP) (cynomolgus monkey) (n=2) as a bolusintravenous injection (IV) over approximately 30 seconds using ahypodermic needle, which may be attached to a syringe/abbocath orbutterfly if needed. The NHP were administered a single modified mRNA IVdose of 0.05 mg/kg of EPO or G-CSF or 0.005 mg/kg of EPO in a dosevolume of 0.5 mL/kg. The NHPs were bled 5-6 days before dosing with themodified mRNA LNP formulations to determine protein expression in theserum and a baseline complete blood count. After administration with themodified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours todetermined protein expression. At 24 and 72 hours after administrationthe complete blood count of the NHP was also determined. Proteinexpression of G-CSF and EPO was determined by ELISA. Urine from the NHPswas collected over the course of the entire experiment and analyzed toevaluate clinical safety. Samples were collected from the NHPs afterthey were administered with G-CSF or EPO modified mRNA formulations todetermine protein expression using ELISA. Clinical chemistry,hematology, urinalysis and cytokines of the non-human primates were alsoanalyzed.

As shown in Table 35, EPO protein expression in the NHPs administered0.05 mg/kg is detectable out to 72 hours and the 0.005 mg/kg dosing ofthe EPO formulation is detectable out to 48 hours. In Table 35, the “<”means less than a given value. G-CSF protein expression was seen out to24 hours after administration with the modified mRNA formulation.Preliminarily, there was an increase in granulocytes and reticulocyteslevels seen in the NHP after administration with the modified mRNAformulations.

TABLE 35 Protein Expression in Non-Human Primates Average Female MaleSerum Dose NHP Serum NHP Serum Conen- Modified (mg/ ConcentrationConcentration tration mRNA kg) Time (pg/ml) (pg/ml) (pg/ml) G-CSF 0.05Pre-bleed 0 0 0  8 hours 3289 1722 2,506 24 hours 722 307 515 48 hours 00 0 72 hours 0 0 0 EPO 0.05 Pre-bleed 0 0 0  8 hours 19,858 7,072 13,46524 hours 18,178 4,913 11,546 48 hours 5,291 498 2,895 72 hours 744 60402 EPO 0.005 Pre-bleed 0 0 0  8 hours 523 250 387 24 hours 302 113 20848 hours <7.8 <7.8 <7.8 72 hours 0 0 0

Example 22 Non-Human Primate In Vivo Study for G-CSF and EPO

LNP formulations, shown in Table 31 (above), were administered tonon-human primates (NHP) (cynomolgus monkey) (n=2) as intravenousinjection (IV). The NHP were administered a single modified mRNA IV doseof 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg of G-CSF or EPO in a dose volumeof 0.5 mL/kg. The NHPs were bled before dosing with the modified mRNALNP formulations to determine protein expression in the serum and abaseline complete blood count. After administration with the G-CSFmodified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours todetermined protein expression. After administration with the EPOmodified mRNA formulation the NHP were bled at 8, 24, 48, 72 hours and 7days to determined protein expression.

Samples collected from the NHPs after they were administered with G-CSFor EPO modified mRNA formulations were analyzed by ELISA to determineprotein expression. Neutrophil and reticulocyte count was alsodetermined pre-dose, 24 hours, 3 days, 7 days, 14 days and 18 days afteradministration of the modified G-CSF or EPO formulation.

As shown in Table 36, G-CSF protein expression was not detected beyond72 hours. In Table 36, “<39” refers to a value below the lower limit ofdetection of 39 pg/ml.

TABLE 36 G-CSF Protein Expression Female NHP Male NHP Serum G-CSF SerumG-CSF Modified Concentration Concentration mRNA Dose (mg/kg) Time(pg/ml) (pg/ml) G-CSF 0.5 Pre-bleed <39 <39  8 hours 43,525 43,594 24hours 11,374 3,628 48 hours 1,100 833 72 hours <39 306 G-CSF 0.05Pre-bleed <39 <39  8 hours 3,289 1,722 24 hours 722 307 48 hours <39 <3972 hours <39 <39 G-CSF 0.005 Pre-bleed <39 <39  8 hours 559 700 24 hours155 <39 48 hours <39 <39 72 hours <39 <39

As shown in Table 37, EPO protein expression was not detected beyond 7days. In Table 37, “<7.8” refers to a value below the lower limit ofdetection of 7.8 pg/ml.

TABLE 37 EPO Protein Expression Female NHP Serum EPO Male NHP SerumModified Dose Concentration EPO Concentration mRNA (mg/kg) Time (pg/ml)(pg/ml) EPO 0.5 Pre-bleed <7.8 <7.8 8 hours 158,771 119,086 24 hours133,978 85,825 48 hours 45,250 64,793 72 hours 15,097 20,407 7 days <7.8<7.8 EPO 0.05 Pre-bleed <7.8 <7.8 8 hours 19,858 7,072 24 hours 18,1874,913 48 hours 5,291 498 72 hours 744 60 7 days <7.8 <7.8 EPO 0.005Pre-bleed <7.8 <7.8 8 hours 523 250 24 hours 302 113 48 hours 11 29 72hours <7.8 <7.8 7 days <7.8 <7.8

As shown in Table 38, there was an increase in neutrophils in all G-CSFgroups relative to pre-dose levels.

TABLE 38 Pharmacologic Effect of G-CSF mRNA in NHP Male NHP Male NHP(G-CSF) Female NHP (EPO) Female NHP Dose Neutro- (G-CSF) Neutro- (EPO)(mg/ phils Neutrophils phils Neutrophils kg) Time (10⁹/L) (10⁹/L)(10⁹/L) (10⁹/L) 0.5 Pre-dose 1.53 1.27 9.72 1.82 24 hours 14.92 13.967.5 11.85 3 days 9.76 13.7 11.07 5.22 7 days 2.74 3.81 11.8 2.85 14/18days 2.58 1.98 7.16 2.36 0.05 Pre-dose 13.74 3.05 0.97 2.15 24 hours19.92 29.91 2.51 2.63 3 days 7.49 10.77 1.73 4.08 7 days 4.13 3.8 1.232.77 14/18 days 3.59 1.82 1.53 1.27 0.005 Pre-dose 1.52 2.54 5.46 5.9624 hours 16.44 8.6 5.37 2.59 3 days 3.74 1.78 6.08 2.83 7 days 7.28 2.273.51 2.23 14/18 days 4.31 2.28 1.52 2.54

As shown in Table 39, there was an increase in reticulocytes in all EPOgroups 3 days to 14/18 days after dosing relative to reticulocyte levels24 hours after dosing.

TABLE 39 Pharmacologic Effect of EPO mRNA on Neutrophil Count Male NHPMale NHP (G-CSF) Female NHP (EPO) Female NHP Dose Neutro- (G-CSF)Neutro- (EPO) (mg/ phils Neutrophils phils Neutrophils kg) Time (10¹²/L)(10¹²/L) (10¹²/L) (10¹²/L) 0.5 Pre-dose 0.067 0.055 0.107 0.06 24 hours0.032 0.046 0.049 0.045 3 days 0.041 0.017 0.09 0.064 7 days 0.009 0.0210.35 0.367 14/18 days 0.029 0.071 0.066 0.071 0.05 Pre-dose 0.055 0.0490.054 0.032 24 hours 0.048 0.046 0.071 0.04 3 days 0.101 0.061 0.1020.105 7 days 0.157 0.094 0.15 0.241 14/18 days 0.107 0.06 0.067 0.0550.005 Pre-dose 0.037 0.06 0.036 0.052 24 hours 0.037 0.07 0.034 0.061 3days 0.037 0.054 0.079 0.118 7 days 0.046 0.066 0.049 0.087 14/18 days0.069 0.057 0.037 0.06

As shown in Tables 40-42, the administration of EPO modified RNA had aneffect on other erythropoetic parameters including hemoglobin (HGB),hematocrit (HCT) and red blood cell (RBC) count.

TABLE 40 Pharmacologic Effect of EPO mRNA on Hemoglobin Male NHP MaleNHP Dose (G-CSF) Female NHP (EPO) Female NHP (mg/ HGB (G-CSF) HGB (EPO)kg) Time (g/L) HGB (g/L) (g/L) HGB (g/L) 0.5 Pre-dose 133 129 134 123 24hours 113 112 127 108 3 days 118 114 126 120 7 days 115 116 140 13414/18 days 98 113 146 133 0.05 Pre-dose 137 129 133 133 24 hours 122 117123 116 3 days 126 115 116 120 7 days 126 116 126 121 14/18 days 134 123133 129 0.005 Pre-dose 128 129 132 136 24 hours 117 127 122 128 3 days116 127 125 130 7 days 116 129 119 127 14/18 days 118 129 128 129

TABLE 41 Pharmacologic Effect of EPO mRNA on Hematocrit Male NHP MaleNHP Dose (G-CSF) Female NHP (EPO) Female NHP (mg/ HCT (G-CSF) HCT (EPO)kg) Time (L/L) HCT (L/L) (L/L) HCT (L/L) 0.5 Pre-dose 0.46 0.43 0.44 0.424 hours 0.37 0.38 0.4 0.36 3 days 0.39 0.38 0.41 0.39 7 days 0.39 0.380.45 0.45 14/18 days 0.34 0.37 0.48 0.46 0.05 Pre-dose 0.44 0.44 0.450.43 24 hours 0.39 0.4 0.43 0.39 3 days 0.41 0.39 0.38 0.4 7 days 0.420.4 0.45 0.41 14/18 days 0.44 0.4 0.46 0.43 0.005 Pre-dose 0.42 0.420.48 0.45 24 hours 0.4 0.42 0.42 0.43 3 days 0.4 0.41 0.44 0.42 7 days0.39 0.42 0.41 0.42 14/18 days 0.41 0.42 0.42 0.42

TABLE 42 Pharmacologic Effect of EPO mRNA on Red Blood Cells Male NHPFemale NHP Male NHP Female NHP Dose (G-CSF) (G-CSF) (EPO) (EPO) (mg/ RBCRBC RBC RBC kg) Time (10¹²/L) (10¹²/L) (10¹²/L) (10¹²/L) 0.5 Pre-dose5.57 5.57 5.43 5.26 24 hours 4.66 4.96 5.12 4.69 3 days 4.91 4.97 5.135.15 7 days 4.8 5.04 5.55 5.68 14/18 days 4.21 4.92 5.83 5.72 0.05Pre-dose 5.68 5.64 5.57 5.84 24 hours 4.96 5.08 5.25 5.18 3 days 5.135.04 4.81 5.16 7 days 5.17 5.05 5.37 5.31 14/18 days 5.43 5.26 5.57 5.570.005 Pre-dose 5.67 5.36 6.15 5.72 4 hours 5.34 5.35 5.63 5.35 3 days5.32 5.24 5.77 5.42 7 days 5.25 5.34 5.49 5.35 14/18 days 5.37 5.34 5.675.36

As shown in Tables 43 and 44, the administration of modified RNA had aneffect on serum chemistry parameters including alanine transaminase(ALT) and aspartate transaminase (AST).

TABLE 43 Pharmacologic Effect of EPO mRNA on Alanine Transaminase MaleNHP Male NHP (G-CSF) Female NHP (EPO) Female NHP Dose ALT (G-CSF) ALT(EPO) (mg/kg) Time (U/L) ALT (U/L) (U/L) ALT (U/L) 0.5 Pre-dose 29 21650 31 2 days 63 209 98 77 4 days 70 98 94 87 7 days 41 149 60 59 14days  43 145 88 44 0.05 Pre-dose 58 53 56 160 2 days 82 39 95 254 4 days88 56 70 200 7 days 73 73 64 187 14 days  50 31 29 216 0.005 Pre-dose 4351 45 45 2 days 39 32 62 48 4 days 48 58 48 50 7 days 29 55 21 48 14days  44 46 43 51

TABLE 44 Pharmacologic Effect of EPO mRNA on Aspartate Transaminase MaleNHP Male NHP (G-CSF) Female NHP (EPO) Female NHP Dose AST (G-CSF) AST(EPO) (mg/kg) Time (U/L) AST (U/L) (U/L) AST (U/L) 0.5 Pre-dose 32 47 5920 2 days 196 294 125 141 4 days 67 63 71 60 7 days 53 68 56 47 14 days 47 67 82 44 0.05 Pre-dose 99 33 74 58 2 days 95 34 61 80 4 days 69 42 4894 7 days 62 52 53 78 14 days  59 20 32 47 0.005 Pre-dose 35 54 39 40 2days 70 34 29 25 4 days 39 36 43 55 7 days 28 31 55 31 14 days  39 20 3554

As shown in Table 45, the administration of modified RNA cause anincrease in cytokines, interferon-alpha (IFN-alpha) after administrationof modified mRNA.

TABLE 45 Pharmacologic Effect of EPO mRNA on Alanine Transaminase MaleNHP Female NHP Male NHP Female NHP Dose (G-CSF) (G-CSF) (EPO) (EPO) (mg/IFN-alpha IFN-alpha IFN-alpha IFN-alpha kg) Time (pg/mL) (pg/mL) (pg/mL)(pg/mL) 0.5 Pre-dose 0 0 0 0 Day 1 + 8 hr 503.8 529.2 16.79 217.5 4 days0 0 0 0 0.05 Pre-dose 0 0 0 0 Day 1 + 8 hr 0 0 0 0 4 days 0 0 0 0 0.005Pre-dose 0 0 0 0 Day 1 + 8 hr 0 0 0 0 4 days 0 0 0 0

Example 23 Study of Intramuscular and/or Subcutaneous Administration inNon-Human Primates

Formulations containing modified EPO mRNA (SEQ ID NO: 9; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) or G-CSF mRNA (SEQ IDNO: 6; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) in saline were administered to non-human primates(Cynomolgus monkey) (NHP) intramuscularly (IM) or subcutaneously (SC).The single modified mRNA dose of 0.05 mg/kg or 0.005 mg/kg was in a dosevolume of 0.5 mL/kg. The non-human primates are bled 5-6 days prior todosing to determine serum protein concentration and a baseline completeblood count. After administration with the modified mRNA formulation theNHP are bled at 8 hours, 24 hours, 48 hours, 72 hours, 7 days and 14days to determined protein expression. Protein expression of G-CSF andEPO is determined by ELISA. At 24 hours, 72 hours, 7 days and 14 daysafter administration the complete blood count of the NHP is alsodetermined. Urine from the NHPs is collected over the course of theentire experiment and analyzed to evaluate clinical safety. Tissue nearthe injection site is also collected and analyzed to determine proteinexpression.

Example 24 Modified mRNA Trafficking

In order to determine localization and/or trafficking of the modifiedmRNA, studies may be performed as follows.

LNP formulations of siRNA and modified mRNA are formulated according tomethods known in the art and/or described herein. The LNP formulationsmay include at least one modified mRNA which may encode a protein suchas G-CSF, EPO, Factor VII, and/or any protein described herein. Theformulations may be administered locally into muscle of mammals usingintramuscular or subcutaneous injection. The dose of modified mRNA andthe size of the LNP may be varied to determine the effect on traffickingin the body of the mammal and/or to assess the impact on a biologicreaction such as, but not limited to, inflammation. The mammal may bebled at different time points to determine the expression of proteinencoded by the modified mRNA administered present in the serum and/or todetermine the complete blood count in the mammal.

For example, modified mRNA encoding Factor VII, expressed in the liverand secreted into the serum, may be administered intramuscularly and/orsubcutaneously. Coincident or prior to modified mRNA administration,siRNA is administered to knock out endogenous Factor VII. Factor VIIarising from the intramuscular and/or subcutaneous injection of modifiedmRNA is administered is measured in the blood. Also, the levels ofFactor VII is measured in the tissues near the injection site. If FactorVII is expressed in blood then there is trafficking of the modifiedmRNA. If Factor VII is expressed in tissue and not in the blood thanthere is only local expression of Factor VII.

Example 25 Formulations of Multiple Modified mRNA

LNP formulations of modified mRNA are formulated according to methodsknown in the art and/or described herein. The LNP formulations mayinclude at least one modified mRNA which may encode a protein such asG-CSF, EPO, thrombopoietin and/or any protein described herein or knownin the art. The at least one modified mRNA may include 1, 2, 3, 4 or 5modified mRNA molecules. The formulations containing at least onemodified mRNA may be administered intravenously, intramuscularly orsubcutaneously in a single or multiple dosing regimens. Biologicalsamples such as, but not limited to, blood and/or serum may be collectedand analyzed at different time points before and/or after administrationof the at least one modified mRNA formulation. An expression of aprotein in a biological sample of 50-200 pg/ml after the mammal has beenadministered a formulation containing at least one modified mRNAencoding said protein would be considered biologically effective.

Example 26 Polyethylene Glycol Ratio Studies

A. Formulation and Characterization of PEG LNPs

Lipid nanoparticles (LNPs) were formulated using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified G-CSF mRNA (SEQ ID NO: 6; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine). The molar ratio ranges of theformulations are shown in Table 46.

TABLE 46 Molar Ratios DLin-KC2-DMA DSPC Cholesterol PEG-c-DOMG MolePercent 50.0 10.0 37-38.5 1.5-3 (mol %)

Two types of PEG lipid, 1,2-Dimyristoyl-sn-glycerol, methoxypolyethyleneGlycol (PEG-DMG, NOF Cat # SUNBRIGHT® GM-020) and1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG, NOF Cat# SUNBRIGHT® GS-020), were tested at 1.5 or 3.0 mol %. After theformation of the LNPs and the encapsulation of the modified G-CSF mRNA,the LNP formulations were characterized by particle size, zeta potentialand encapsulation percentage and the results are shown in Table 47.

TABLE 47 Characterization of LNP Formulations Formulation No. NPA-071-1NPA-072-1 NPA-073-1 NPA-074-1 Lipid PEG-DMG PEG-DMG PEG-DSA PEG-DSA 1.5%3% 1.5% 3% Mean Size 95 nm 85 nm 95 nm 75 nm PDI: 0.01 PDI: 0.06 PDI:0.08 PDI: 0.08 Zeta at pH 7.4 −1.1 mV −2.6 mV 1.7 mV 0.7 mVEncapsulation 88% 89% 98% 95% (RiboGreen)

B. In Vivo Screening of PEG LNPs

Formulations of the PEG LNPs described in Table 40 were administered tomice (n=5) intravenously at a dose of 0.5 mg/kg. Serum was collectedfrom the mice at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 8days after administration of the formulation. The serum was analyzed byELISA to determine the protein expression of G-CSF and the expressionlevels are shown in Table 48. LNP formulations using PEG-DMG gavesubstantially higher levels of protein expression than LNP formulationswith PEG-DSA.

TABLE 48 Protein Expression Formulation Protein Expression Lipid No.Time (pg/ml) PEG-DMG, NPA-071-1 2 hours 114,102 1.5% 8 hours 357,944 24hours 104,832 48 hours 6,697 72 hours 980 8 days 0 PEG-DMG, 3% NPA-072-12 hours 154,079 8 hours 354,994 24 hours 164,311 48 hours 13,048 72hours 1,182 8 days 13 PEG-DSA, 1.5% NPA-073-1 2 hours 3,193 8 hours6,162 24 hours 446 48 hours 197 72 hours 124 8 days 5 PEG-DSA, 3%NPA-074-1 2 hours 259 8 hours 567 24 hours 258 48 hours 160 72 hours 3288 days 33

Example 27 Cationic Lipid Formulation Studies

A. Formulation and Characterization of Cationic Lipid Nanoparticles

Lipid nanoparticles (LNPs) were formulated using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified mRNA. The final lipid molar ratio ranges of cationic lipid,DSPC, cholesterol and PEG-c-DOMG are outlined in Table 49.

TABLE 49 Molar Ratios Cationic Lipid DSPC Cholesterol PEG-c-DOMG MolePercent 50.0 10.0 38.5 1.5 (mol %)

A 25 mM lipid solution in ethanol and modified RNA in 50 mM citrate at apH of 3 were mixed to create spontaneous vesicle formation. The vesicleswere stabilized in ethanol before the ethanol was removed and there wasa buffer exchange by dialysis. The LNPs were then characterized byparticle size, zeta potential, and encapsulation percentage. Table 50describes the characterization of LNPs encapsulating EPO modified mRNA(SEQ ID NO: 9 polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) or G-CSF modified mRNA (SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) using DLin-MC3-DMA,DLin-DMA or C12-200 as the cationic lipid.

TABLE 50 Characterization of Cationic Lipid Formulations Formulation No.NPA- NPA- NPA- NPA- NPA- NPA- 071-1 072-1 073-1 074-1 075-1 076-1 LipidDLin- DLin- DLin- DLin- C12- C12- MC3- MC3- DMA DMA 200 200 DMA DMAModified EPO G-CSF EPO G-CSF EPO G-CSF RNA Mean Size 89 nm 96 nm 70 nm73 nm 97 nm 103 nm PDI: PDI: PDI: PDI: PDI: PDI: 0.07 0.08 0.04 0.060.05 0.09 Zeta at pH 7.4 −1.1 mV −1.4 mV −1.6 mV −0.4 mV 1.4 mV 0.9 mVEncapsulation 100% 100% 99% 100% 88% 98% (RiboGreen)

B. In Vivo Screening of Cationic LNP Formulations

Formulations of the cationic lipid formulations described in Table 42were administered to mice (n=5) intravenously at a dose of 0.5 mg/kg.Serum was collected from the mice at 2 hours, 24 hours, 72 hours and/or7 days after administration of the formulation. The serum was analyzedby ELISA to determine the protein expression of EPO or G-CSF and theexpression levels are shown in Table 51.

TABLE 51 Protein Expression Protein Modified Formulation Expression mRNANo. Time (pg/ml) EPO NPA-071-1 2 hours 304,190.0 24 hours 166,811.5 72hours 1,356.1 7 days 20.3 EPO NPA-073-1 2 hours 73,852.0 24 hours75,559.7 72 hours 130.8 EPO NPA-075-1 2 hours 413,010.2 24 hours56,463.8 G-CSF NPA-072-1 2 hours 62,113.1 24 hours 53,206.6 G-CSFNPA-074-1 24 hours 25,059.3 G-CSF NPA-076-1 2 hours 219,198.1 24 hours8,470.0

Toxcity was seen in the mice administered the LNPs formulations with thecationic lipid C12-200 (NPA-075-1 and NPA-076-1) and they weresacrificed at 24 hours because they showed symptoms such as scrubby fur,cowering behavior and weight loss of greater than 10%. C12-200 wasexpected to be more toxic but also had a high level of expression over ashort period. The cationic lipid DLin-DMA (NPA-073-1 and NPA-074-1) hadthe lowest expression out of the three cationic lipids tested.DLin-MC3-DMA (NPA-071-1 and NPA-072-1) showed good expression up to daythree and was above the background sample out to day 7 for EPOformulations.

Example 28 Method of Screening for Protein Expression

A. Electrospray Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or4 mass analyzers. A biologic sample may also be analyzed using a tandemESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for matrix-assisted laser desorption/ionization(MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by modified RNA,may be treated with a trypsin enzyme to digest the proteins containedwithin. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g. water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g. detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 29 LNP In Vivo Studies

mCherry mRNA (SEQ ID NO: 14; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) was formulated as a lipidnanoparticle (LNP) using the syringe pump method. The LNP was formulatedat a 20:1 weight ratio of total lipid to modified mRNA with a finallipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). The mCherry formulation,listed in Table 52, was characterized by particle size, zeta potential,and encapsulation.

TABLE 52 mCherry Formulation Formulation # NPA-003-5 Modified mRNAmCherry Mean size 105 nm PDI: 0.09 Zeta at pH 7.4 1.8 mV Encaps. 100%(RiboGr)

The LNP formulation was administered to mice (n=5) intravenously at amodified mRNA dose of 100 ug. Mice were sacrificed at 24 hrs afterdosing. The liver and spleen from the mice administered with mCherrymodified mRNA formulations were analyzed by immunohistochemistry (IHC),western blot, or fluorescence-activated cell sorting (FACS).

Histology of the liver showed uniform mCherry expression throughout thesection, while untreated animals did not express mCherry. Western blotswere also used to confirm mCherry expression in the treated animals,whereas mCherry was not detected in the untreated animals. Tubulin wasused as a control marker and was detected in both treated and untreatedmice, indicating that normal protein expression in hepatocytes wasunaffected.

FACS and IHC were also performed on the spleens of mCherry and untreatedmice. All leukocyte cell populations were negative for mCherryexpression by FACS analysis. By IHC, there were also no observabledifferences in the spleen in the spleen between mCherry treated anduntreated mice.

Example 30 Syringe Pump in Vivo Studies

mCherry modified mRNA is formulated as a lipid nanoparticle (LNP) usingthe syringe pump method. The LNP is formulated at a 20:1 weight ratio oftotal lipid to modified mRNA with a final lipid molar ratio of50:10:38.5:1.5 (DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). The mCherryformulation is characterized by particle size, zeta potential, andencapsulation.

The LNP formulation is administered to mice (n=5) intravenously at amodified mRNA dose of 10 or 100 ug. Mice are sacrificed at 24 hrs afterdosing. The liver and spleen from the mice administered with mCherrymodified mRNA formulations are analyzed by immunohistochemistry (IHC),western blot, and/or fluorescence-activated cell sorting (FACS).

Example 31 In Vitro and In Vivo Expression

A. In Vitro Expression in Human Cells Using Lipidoid Formulations

The ratio of mmRNA to lipidoid used to test for in vitro transfection istested empirically at different lipidoid:mmRNA ratios. Previous workusing siRNA and lipidoids have utilized 2.5:1, 5:1, 10:1, and 15:1lipidoid:siRNA wt:wt ratios. Given the longer length of mmRNA relativeto siRNA, a lower wt:wt ratio of lipidoid to mmRNA may be effective. Inaddition, for comparison mmRNA were also formulated using RNAIMAX™(Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison,Wis.) cationic lipid delivery vehicles. The ability oflipidoid-formulated Luciferase (IVT cDNA sequence as shown in SEQ ID NO:15; mRNA sequence shown in SEQ ID NO: 16, polyA tail of approximately160 nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site), green fluorescent protein (GFP) (IVT cDNA wild-typesequence is shown in SEQ ID NO: 17; mRNA sequence shown in SEQ ID NO:18, polyA tail of approximately 160 nucleotides not shown in sequence,5′ cap, Cap1), G-CSF (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1), andEPO mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) toexpress the desired protein product can be confirmed by luminescence forluciferase expression, flow cytometry for GFP expression, and by ELISAfor G-CSF and Erythropoietin (EPO) secretion.

B. In Vivo Expression Following Intravenous Injection

Systemic intravenous administration of the formulations are createdusing various different lipidoids including, but not limited to,98N12-5, C12-200, and MD1.

Lipidoid formulations containing mmRNA are injected intravenously intoanimals. The expression of the modified mRNA (mmRNA)-encoded proteinsare assessed in blood and/or other organs samples such as, but notlimited to, the liver and spleen collected from the animal. Conductingsingle dose intravenous studies will also allow an assessment of themagnitude, dose responsiveness, and longevity of expression of thedesired product.

In one embodiment, lipidoid based formulations of 98N12-5, C12-200, MD1and other lipidoids, are used to deliver luciferase, green fluorescentprotein (GFP), mCherry fluorescent protein, secreted alkalinephosphatase (sAP), human G-CSF, human Factor IX, or human Erythropoietin(EPO) mmRNA into the animal. After formulating mmRNA with a lipid, asdescribed previously, animals are divided into groups to receive eithera saline formulation, or a lipidoid-formulation which contains one of adifferent mmRNA selected from luciferase, GFP, mCherry, sAP, humanG-CSF, human Factor IX, and human EPO. Prior to injection into theanimal, mmRNA-containing lipidoid formulations are diluted in PBS.Animals are then administered a single dose of formulated mmRNA rangingfrom a dose of 10 mg/kg to doses as low as 1 ng/kg, with a preferredrange to be 10 mg/kg to 100 ng/kg, where the dose of mmRNA depends onthe animal body weight such as a 20 gram mouse receiving a maximumformulation of 0.2 ml (dosing is based no mmRNA per kg body weight).After the administration of the mmRNA-lipidoid formulation, serum,tissues, and/or tissue lysates are obtained and the level of themmRNA-encoded product is determined at a single and/or a range of timeintervals. The ability of lipidoid-formulated Luciferase, GFP, mCherry,sAP, G-CSF, Factor IX, and EPO mmRNA to express the desired proteinproduct is confirmed by luminescence for the expression of Luciferase,flow cytometry for the expression of GFP and mCherry expression, byenzymatic activity for sAP, or by ELISA for the section of G-CSF, FactorIX and/or EPO.

Further studies for a multi-dose regimen are also performed to determinethe maximal expression of mmRNA, to evaluate the saturability of themmRNA-driven expression (by giving a control and active mmRNAformulation in parallel or in sequence), and to determine thefeasibility of repeat drug administration (by giving mmRNA in dosesseparated by weeks or months and then determining whether expressionlevel is affected by factors such as immunogenicity). An assessment ofthe physiological function of proteins such as G-CSF and EPO are alsodetermined through analyzing samples from the animal tested anddetecting increases in granulocyte and red blood cell counts,respectively. Activity of an expressed protein product such as FactorIX, in animals can also be assessed through analysis of Factor IXenzymatic activity (such as an activated partial thromboplastin timeassay) and effect of clotting times.

C. In Vitro Expression Following Intramuscular and/or SubcutaneousInjection

The use of lipidoid formulations to deliver oligonucleotides, includingmRNA, via an intramuscular route or a subcutaneous route of injectionneeds to be evaluated as it has not been previously reported.Intramuscular and/or subcutaneous injection of mmRNA are evaluated todetermine if mmRNA-containing lipidoid formulations are capabable toproduce both localized and systemic expression of a desired proteins.

Lipidoid formulations of 98N12-5, C12-200, and MD1 containing mmRNAselected from luciferase, green fluorescent protein (GFP), mCherryfluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF,human factor IX, or human Erythropoietin (EPO) mmRNA are injectedintramuscularly and/or subcutaneously into animals. The expression ofmmRNA-encoded proteins are assessed both within the muscle orsubcutaneous tissue and systemically in blood and other organs such asthe liver and spleen. Single dose studies allow an assessment of themagnitude, dose responsiveness, and longevity of expression of thedesired product.

Animals are divided into groups to receive either a saline formulationor a formulation containing modified mRNA. Prior to injectionmmRNA-containing lipidoid formulations are diluted in PBS. Animals areadministered a single intramuscular dose of formulated mmRNA rangingfrom 50 mg/kg to doses as low as 1 ng/kg with a preferred range to be 10mg/kg to 100 ng/kg. A maximum dose for intramuscular administration, fora mouse, is roughly 1 mg mmRNA or as low as 0.02 ng mmRNA for anintramuscular injection into the hind limb of the mouse. Forsubcutaneous administration, the animals are administered a singlesubcutaneous dose of formulated mmRNA ranging from 400 mg/kg to doses aslow as 1 ng/kg with a preferred range to be 80 mg/kg to 100 ng/kg. Amaximum dose for subcutaneous administration, for a mouse, is roughly 8mg mmRNA or as low as 0.02 ng mmRNA.

For a 20 gram mouse the volume of a single intramuscular injection ismaximally 0.025 ml and a single subcutaneous injection is maximally 0.2ml. The optimal dose of mmRNA administered is calculated from the bodyweight of the animal. At various points in time points following theadministration of the mmRNA-lipidoid, serum, tissues, and tissue lysatesis obtained and the level of the mmRNA-encoded product is determined.The ability of lipidoid-formulated luciferase, green fluorescent protein(GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP),human G-CSF, human factor IX, or human Erythropoietin (EPO) mmRNA toexpress the desired protein product is confirmed by luminescence forluciferase expression, flow cytometry for GFP and mCherry expression, byenzymatic activity for sAP, and by ELISA for G-CSF, Factor IX andErythropoietin (EPO) secretion.

Additional studies for a multi-dose regimen are also performed todetermine the maximal expression using mmRNA, to evaluate thesaturability of the mmRNA-driven expression (achieved by giving acontrol and active mmRNA formulation in parallel or in sequence), and todetermine the feasibility of repeat drug administration (by giving mmRNAin doses separated by weeks or months and then determining whetherexpression level is affected by factors such as immunogenicity). Studiesutilizing multiple subcutaneous or intramuscular injection sites at onetime point, are also utilized to further increase mmRNA drug exposureand improve protein production. An assessment of the physiologicalfunction of proteins, such as GFP, mCherry, sAP, human G-CSF, humanfactor IX, and human EPO, are determined through analyzing samples fromthe tested animals and detecting a change in granulocyte and/or redblood cell counts. Activity of an expressed protein product such asFactor IX, in animals can also be assessed through analysis of Factor IXenzymatic activity (such as an activated partial thromboplastin timeassay) and effect of clotting times.

Example 32 In Vivo Delivery Using Lipoplexes

A. Human EPO Modified RNA Lipoplex

A formulation containing 100 μg of modified human erythropoietin (EPO)mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1) (EPO; fullymodified 5-methylcytosine; N1-methylpseudouridine) was lipoplexed with30% by volume of RNAIMAX™ (Lipoplex-h-Epo-46; Generation 2 or Gen2) in50-70 uL delivered intramuscularly to four C57/BL6 mice. Other groupsconsisted of mice receiving an injection of the lipoplexed modifiedluciferase mRNA (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO:15; mRNA sequence shown in SEQ ID NO: 16, polyA tail of approximately160 nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) which served as a control containing 100 μg of modifiedluciferase mRNA was lipoplexed with 30% by volume of RNAiMAX™ or micereceiving an injection of the formulation buffer as negative control ata dose volume of 65 ul. 13 hours after the intramuscular injection,serum was collected from each mouse to measure the amount of human EPOprotein in the mouse serum by human EPO ELISA and the results are shownin Table 53.

TABLE 53 Human EPO Production (IM Injection Route) Formualtion Mouse #1Mouse #2 Mouse #3 Mouse #4 Average Lipoplex-h- 189.8 92.55 409.5 315.95251.95 Epo-46 Lipoplex-Luc 0 0 0 0 0 Formulation 0 0 0 0 0 Buffer

B. Human G-CSF Modified RNA Lipoplex

A formulation containing 100 μg of one of two versions of modified humanG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)(G-CSF fully modified with 5-methylcytosine and pseudouridine (G-CSF) orG-CSF fully modified with 5-methylcytosine and N1-methyl-pseudouridine(G-CSF-N1) lipoplexed with 30% by volume of RNAIMAX™ and delivered in150 uL intramuscularly (I.M), in 150 uL subcutaneously (S.C) and in 225uL intravenously (I.V) to C57/BL6 mice.

Three control groups were administered either 100 μg of modifiedluciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 15; mRNA sequenceshown in SEQ ID NO: 16, polyA tail of approximately 160 nucleotides notshown in sequence, 5′ cap, Cap1, fully modified with 5-methylcytosine ateach cytosine and pseudouridine replacement at each uridine site)intramuscularly (Luc-unsp I.M.) or 150 μg of modified luciferase mRNAintravenously (Luc-unsp I.V.) or 150 uL of the formulation bufferintramuscularly (Buffer I.M.). 6 hours after administration of aformulation, serum was collected from each mouse to measure the amountof human G-CSF protein in the mouse serum by human G-CSF ELISA and theresults are shown in Table 54.

These results demonstrate that both 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.V. or I.M. route of administration in a lipoplexformulation.

TABLE 54 Human G-CSF in Serum (I.M., I.V., S.C. Injection Route)Formulation Route G-CSF (pg/ml) G-CSF I.M. 85.6 G-CSF N1 I.M. 40.1 G-CSFS.C. 3.9 G-CSF N1 S.C. 0.0 G-CSF I.V. 31.0 G-CSF N1 I.V. 6.1 Luc-unspI.M. 0.0 Luc-unsp I.V. 0.0 Buffer I.M. 0.0

C. Human G-CSF Modified RNA Lipoplex Comparison

A formulation containing 100 μg of either modified human G-CSF mRNAlipoplexed with 30% by volume of RNAIMAX™ with a 5-methylcytosine (5mc)and a pseudouridine (ψ) modification (G-CSF-Gen1-Lipoplex), modifiedhuman G-CSF mRNA with a 5mc and ψ modification in saline(G-CSF-Gen1-Saline), modified human G-CSF mRNA with aN1-5-methylcytosine (N1-5mc) and a ψ modification lipoplexed with 30% byvolume of RNAIMAX™ (G-CSF-Gen2-Lipoplex), modified human G-CSF mRNA witha N1-5mc and ψ modification in saline (G-CSF-Gen2-Saline), modifiedluciferase with a 5mc and ψ modification lipoplexed with 30% by volumeof RNAIMAX™ (Luc-Lipoplex), or modified luciferase mRNA with a a 5mc andψ modification in saline (Luc-Saline) was delivered intramuscularly(I.M.) or subcutaneously (S.C.) and a control group for each method ofadministration was giving a dose of 80 uL of the formulation buffer (F.Buffer) to C57/BL6 mice. 13 hours post injection serum and tissue fromthe site of injection were collected from each mouse and analyzed byG-CSF ELISA to compare human G-CSF protein levels. The results of thehuman G-CSF protein in mouse serum from the intramuscular administrationand the subcutaneous administration results are shown in Table 55.

These results demonstrate that 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.M. or S.C. route of administration whether in a salineformulation or in a lipoplex formulation. As shown in Table 55,5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNAgenerally demonstrates increased human G-CSF protein production relativeto 5-methylcytosine/pseudouridine modified human G-CSF mRNA.

TABLE 55 Human G-CSF Protein in Mouse Serum G-CSF (pg/ml) FormulationI.M. Injection Route S.C. Injenction Route G-CSF-Gen1-Lipoplex 13.98842.855 GCSF-Gen1-saline 9.375 4.614 GCSF-Gen2-lipoplex 75.572 32.107GCSF-Gen2-saline 20.190 45.024 Luc lipoplex 0 3.754 Luc saline 0.0748 0F. Buffer 4.977 2.156

D. mCherry Modified RNA Lipoplex Comparison

Intramuscular and Subcutaneous Administration

A formulation containing 100 μg of either modified mCherry mRNA (mRNAsequence shown in SEQ ID NO: 7; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) lipoplexed with 30% byvolume of RNAIMAX™ or modified mCherry mRNA in saline is deliveredintramuscularly and subcutaneously to mice. A formulation buffer is alsoadministered to a control group of mice either intramuscularly orsubcutaneously. The site of injection on the mice may be collected 17hours post injection for sectioning to determine the cell type(s)responsible for producing protein.

Intravitreal Administration

A formulation containing 10 μg of either modified mCherry mRNAlipoplexed with RNAIMAX™, modified mCherry mRNA in a formulation buffer,modified luciferase mRNA lipoplexed with RNAMAX™, modified luciferasemRNA in a formulation buffer can be administered by intravitrealinjection (IVT) in rats in a dose volume of 5 μl/eye. A formulationbuffer is also administered by IVT to a control group of rats in a dosevolume of 5 μl/eye. Eyes from treated rats can be collected after 18hours post injection for sectioning and lysating to determine whethermmRNA can be effectively delivered in vivo to the eye and result inprotein production, and to also determine the cell type(s) responsiblefor producing protein in vivo.

Intranasal Administration

A formulation containing 100 μg of either modified mCherry mRNAlipoplexed with 30% by volume of RNAIMAX™, modified mCherry mRNA insaline, modified luciferase mRNA lipoplexed with 30% by volume ofRNAIMAX™ or modified luciferase mRNA in saline is deliveredintranasally. A formulation buffer is also administered to a controlgroup intranasally. Lungs may be collected about 13 hours postinstillation for sectioning (for those receiving mCherry mRNA) orhomogenization (for those receiving luciferase mRNA). These samples willbe used to determine whether mmRNA can be effectively delivered in vivoto the lungs and result in protein production, and to also determine thecell type(s) responsible for producing protein in vivo.

Example 33 In Vivo Delivery Using Varying Lipid Ratios

Modified mRNA was delivered to C57/BL6 mice to evaluate varying lipidratios and the resulting protein expression. Formulations of 100 μgmodified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) lipoplexed with10%, 30% or 50% RNAIMAX™, 100 μg modified luciferase mRNA (IVT cDNAsequence shown in SEQ ID NO: 15; mRNA sequence shown in SEQ ID NO: 16,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site) lipoplexed with 10%, 30%or 50% RNAIMAX™ or a formulation buffer were administeredintramuscularly to mice in a single 70 μl dose. Serum was collected 13hours post injection to undergo a human EPO ELISA to determine the humanEPO protein level in each mouse. The results of the human EPO ELISA,shown in Table 56, show that modified human EPO expressed in the muscleis secreted into the serum for each of the different percentage ofRNAIMAX™.

TABLE 56 Human EPO Protein in Mouse Serum (IM Injection Route)Formulation EPO (pg/ml) Epo + 10% RNAiMAX 11.4 Luc + 10% RNAiMAX 0 Epo +30% RNAiMAX 27.1 Luc + 30% RNAiMAX 0 Epo + 50% RNAiMAX 19.7 Luc + 50%RNAiMAX 0 F. Buffer 0

Example 34 Intramuscular and Subcutaneous in Vivo Delivery in Mammals

Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) formulated informulation buffer was delivered to either C57/BL6 mice orSprague-Dawley rats to evaluate the dose dependency on human EPOproduction. Rats were intramuscularlly injected with 50 μl of themodified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) (IVTcDNA sequence shown in SEQ ID NO: 15; mRNA sequence shown in SEQ ID NO:16, polyA tail of approximately 160 nucleotides not shown in sequence,5′ cap, Cap1, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site) or the formulationbuffer (F.Buffer) as described in the dosing chart Table 57.

Mice were intramuscularly or subcutaneously injected with 50 μl of themodified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) or theformulation buffer (F.Buffer) as described in the dosing chart Table 58.13 hours post injection blood was collected and serum was analyzed todetermine the amount human EPO for each mouse or rat. The average andgeometric mean in pg/ml for the rat study are also shown in Table 57.

TABLE 57 Rat Study Geometric- Avg. mean Group Dose pg/ml pg/ml h-EPO G#1150 μg 67.7 67.1 h-EPO G#2 100 μg 79.4 66.9 h-EPO G#3  50 μg 101.5 85.4h-EPO G#4  10 μg 46.3 31.2 h-EPO G#5  1 μg 28.7 25.4 Luc G#6 100 μg 24.522.4 F. Buffer G#7 — 18.7 18.5

TABLE 58 Mouse Study Average Level in serum Route Treatment Group Dosepg/ml IM h-EPO 1 100 μg 96.2 IM h-EPO 2  50 μg 63.5 IM h-EPO 3  25 μg18.7 IM h-EPO 4  10 μg 25.9 IM h-EPO 5  1 μg 2.6 IM Luc 6 100 μg 0 IM F.Buffer 7 — 1.0 SC h-EPO 1 100 μg 72.0 SC Luc 2 100 μg 26.7 SC F. Buffer3 — 17.4

Example 35 Duration of Activity after Intramuscular in Vivo Delivery

Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) formulated informulation buffer was delivered to Sprague-Dawley rats to determine theduration of the dose response. Rats were intramuscularly injected with50 μl of the modified human EPO mRNA (h-EPO), modified luciferase mRNA(IVT cDNA sequence shown in SEQ ID NO: 15; mRNA sequence shown in SEQ IDNO: 16, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) (Luc) orthe formulation buffer (F.Buffer) as described in the dosing chart Table59. The rats were bled 2, 6, 12, 24, 48 and 72 hours after theintramuscular injection to determine the concentration of human EPO inserum at a given time. The average and geometric mean in pg/ml for thisstudy are also shown in Table 59.

TABLE 59 Dosing Chart Avg. pg/ Geometric- Group Dose ml mean pg/ml h-EPO2 hour 100 μg 59.6 58.2 h-EPO 6 hour 100 μg 68.6 55.8 h-EPO 12 hour 100μg 87.4 84.5 h-EPO 24 hour 100 μg 108.6 95.3 h-EPO 48 hour 100 μg 77.977.0 h-EPO 72 hour 100 μg 80.1 75.8 Luc 24, 48 hour 100 μg 37.2 29.2 and72 F. Buffer 24, 48 hour — 48.9 10.4 and 72

Example 36 Routes of Administration

Studies were performed to investigate split dosing using differentroutes of administration. Studies utilizing multiple subcutaneous orintramuscular injection sites at one time point were designed andperformed to investigate ways to increase mmRNA drug exposure andimprove protein production. In addition to detection of the expressedprotein product, an assessment of the physiological function of proteinswas also determined through analyzing samples from the animal tested.

Surprisingly, it has been determined that split dosing of mmRNA producesgreater protein production and phenotypic responses than those producedby single unit dosing or multi-dosing schemes.

The design of a split dose experiment involved using humanerythropoietin (EPO) mmRNA (mRNA sequence shown in SEQ ID NO: 9; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) or luciferase mmRNA (mRNA sequence shown in SEQ ID NO: 16; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) administered in buffer alone or formulated with 30% lipoplex(RNAIMAX™). The dosing vehicle (formulation buffer) consisted of 150 mMNaCl, 2 mM CaCl₂, 2 mM Na⁺-phosphate (1.4 mM monobasic sodium phosphate;0.6 mM dibasic sodium phosphate), and 0.5 mM EDTA, pH 6.5. The pH wasadjusted using sodium hydroxide and the final solution was filtersterilized. The mmRNA was modified with 5meC at each cytosine andpseudouridine replacement at each uridine site.

mice per group were dosed intramuscularly (I.M.), intravenously (I.V.)or subcutaneously (S.C.) by the dosing chart outlined in Table 60. Serumwas collected 13 hours post injection from all mice, tissue wascollected from the site of injection from the intramuscular andsubcutaneous group and the spleen, liver and kidneys were collected fromthe intravenous group. The results from the intramuscular group and thesubcutaneous group results are shown in Table 61.

TABLE 60 Dosing Chart Total Dosing Group Treatment Route Dose of mmRNADose Vehicle 1 Lipoplex-human EPO mmRNA I.M. 4 × 100 ug + 30% 4 × 70 ulLipoplex Lipoplex 2 Lipoplex-human EPO mmRNA I.M. 4 × 100 ug 4 × 70 ulBuffer 3 Lipoplex-human EPO mmRNA S.C. 4 × 100 ug + 30% 4 × 70 ulLipoplex Lipoplex 4 Lipoplex-human EPO mmRNA S.C. 4 × 100 ug 4 × 70 ulBuffer 5 Lipoplex-human EPO mmRNA I.V. 200 ug + 30% Lipoplex 140 ulLipoplex 6 Lipoplexed-Luciferase mmRNA I.M. 100 ug + 30% Lipoplex 4 × 70ul Lipoplex 7 Lipoplexed-Luciferase mmRNA I.M. 100 ug 4 × 70 ul Buffer 8Lipoplexed-Luciferase mmRNA S.C. 100 ug + 30% Lipoplex 4 × 70 ulLipoplex 9 Lipoplexed-Luciferase mmRNA S.C. 100 ug 4 × 70 ul Buffer 10Lipoplexed-human EPO mmRNA I.V. 200 ug + 30% Lipoplex 140 ul Lipoplex 11Formulation Buffer I.M. 4x multi dosing 4 × 70 ul Buffer

TABLE 61 Human EPO Protein in Mouse Serum (I.M. Injection Route) EPO(pg/ml) Formulation I.M. Injection Route S.C. Injection RouteEpo-Lipoplex 67.115 2.154 Luc-Lipoplex 0 0 Epo-Saline 100.891 11.37Luc-Saline 0 0 Formulation Buffer 0 0

Example 37 Rapidly Eliminated Lipid Nanoparticle (reLNP) Studies

A. Formulation of Modified RNA reLNPs

Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine(DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol(Sigma-Aldrich, Taufkirchen, Germany), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG-c-DOMG) (NOF, Bouwelven, Belgium) are prepared and stored at −20°C. The synthesized lipid is selected from DLin-DMA with an internalester, DLin-DMA with a terminal ester, DLin-MC3-DMA-internal ester, andDLin-MC3-DMA with a terminal ester. The reLNPs are combined to yield amolar ratio of 50:10:38.5:1.5 (reLNP:DSPC:Cholesterol:PEG-c-DOMG).Formulations of the reLNPs and modified mRNA are prepared by combiningthe lipid solution with the modified mRNA solution at total lipid tomodified mRNA weight ratio of 10:1, 15:1, 20:1 and 30:1.

B. Characterization of formulations

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) is used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy is used to determine the concentrationof modified mRNA nanoparticle formulation. After mixing, the absorbancespectrum of the solution is recorded between 230 nm and 330 nm on a DU800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The modified RNA concentration in the nanoparticle formulationis calculated based on the extinction coefficient of the modified RNAused in the formulation and on the difference between the absorbance ata wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.)is used to evaluate the encapsulation of modified RNA by thenanoparticle. The samples are diluted, transferred to a polystyrene 96well plate, then either a TE buffer or a 2% Triton X-100 solution isadded. The plate is incubated and the RIBOGREEN® reagent is diluted inTE buffer, and of this solution is added to each well. The fluorescenceintensity is measured using a fluorescence plate reader (Wallac Victor1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) The fluorescencevalues of the reagent blank are subtracted from each of the samples andthe percentage of free modified RNA is determined by dividing thefluorescence intensity of the intact sample by the fluorescence value ofthe disrupted sample.

C. In Vitro Incubation

Human embryonic kidney epithelial (HEK293) and hepatocellular carcinomaepithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) are seededon 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany) andplates for HEK293 cells are precoated with collagen type1. HEK293 areseeded at a density of about 30,000 and HepG2 are seeded at a density ofabout 35,000 cells per well in 100 μl cell culture medium. Formulationscontaining mCherry mRNA (mRNA sequence shown in SEQ ID NO: 7; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)are added directly after seeding the cells and incubated. The mCherrycDNA with the T7 promoter, 5′ untranslated region (UTR) and 3′ UTR usedin in vitro transcription (IVT) is given in SEQ ID NO: 8.

Cells are harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells are trypsinized with ½ volume Trypsin/EDTA (Biochrom AG,Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples are then submitted toa flow cytometer measurement with an excitation laser and a filter forPE-Texas Red in a LSRII cytometer (Beckton Dickinson GmbH, Heidelberg,Germany). The mean fluorescence intensity (MFI) of all events and thestandard deviation of four independent wells are presented in forsamples analyzed.

D. In Vivo Formulation Studies

Mice are administered intravenously a single dose of a formulationcontaining a modified mRNA and a reLNP. The modified mRNA administeredto the mice is selected from G-CSF (mRNA sequence shown in SEQ ID NO: 6;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), Factor IX (mRNA shown in SEQ ID NO: 10; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) ormCherry (mRNA sequence shown in SEQ ID NO: 7; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1).

The mice are injected with 100 ug, 10 ug or 1 ug of the formulatedmodified mRNA and are sacrificed 8 hours after they are administered theformulation. Serum from the mice administered formulations containinghuman G-CSF modified mRNA are measured by specific G-CSF ELISA and serumfrom mice administered human Factor IX modified RNA is analyzed byspecific factor IX ELISA or chromogenic assay. The liver and spleen fromthe mice administered with mCherry modified mRNA are analyzed byimmunohistochemistry (IHC) or fluorescence-activated cell sorting(FACS). As a control, a group of mice are not injected with anyformulation and their serum and tissue are collected analyzed by ELISA,FACS and/or IHC.

Example 38 In Vitro Transfection of VEGF-A

Human vascular endothelial growth factor-isoform A (VEGF-A) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 19; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1) was transfected viareverse transfection in Human Keratinocyte cells in 24 multi-wellplates. The VEGF-A cDNA with the T7 promoter, 5′ untranslated region(UTR) and 3′ UTR used in in vitro transcription (IVT) is given in SEQ IDNO: 20. Human Keratinocytes cells were grown in EPILIFE® medium withSupplement S7 from Invitrogen (Carlsbad, Calif.) until they reached aconfluence of 50-70%. The cells were transfected with 0, 46.875, 93.75,187.5, 375, 750, and 1500 ng of modified mRNA (mmRNA) encoding VEGF-Awhich had been complexed with RNAIMAX™ from Invitrogen (Carlsbad,Calif.). The RNA:RNAIMAX™ complex was formed by first incubating the RNAwith Supplement-free EPILIFE® media in a 5× volumetric dilution for 10minutes at room temperature. In a second vial, RNAIMAX™ reagent wasincubated with Supplement-free EPILIFE® Media in a 10× volumetricdilution for 10 minutes at room temperature. The RNA vial was then mixedwith the RNAIMAX™ vial and incubated for 20-30 minutes at roomtemperature before being added to the cells in a drop-wise fashion.

The fully optimized mRNA encoding VEGF-A (mRNA sequence shown in SEQ IDNO: 19; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) transfected with the Human Keratinocyte cellsincluded modifications during translation such as natural nucleosidetriphosphates (NTP), pseudouridine at each uridine site and5-methylcytosine at each cytosine site (pseudo-U/5mC), andN1-methyl-pseudouridine at each uridine site and 5-methylcytosine ateach cytosine site (N1-methyl-Pseudo-U/5mC). Cells were transfected withthe mmRNA encoding VEGF-A and secreted VEGF-A concentration (pg/ml) inthe culture medium was measured at 6, 12, 24, and 48 hourspost-transfection for each of the concentrations using an ELISA kit fromInvitrogen (Carlsbad, Calif.) following the manufacturers recommendedinstructions. These data, shown in Table 62, show that modified mRNAencoding VEGF-A is capable of being translated in Human Keratinocytecells and that VEGF-A is transported out of the cells and released intothe extracellular environment.

TABLE 62 VEGF-A Dosing and Protein Secretion 6 hours 12 hours 24 hours48 hours Dose (ng) (pg/ml) (pg/ml) (pg/ml) (pg/ml) VEGF-A DoseContaining Natural NTPs 46.875 10.37 18.07 33.90 67.02 93.75 9.79 20.5441.95 65.75 187.5 14.07 24.56 45.25 64.39 375 19.16 37.53 53.61 88.28750 21.51 38.90 51.44 61.79 1500 36.11 61.90 76.70 86.54 VEGF-A DoseContaining Pseudo-U/5mC 46.875 10.13 16.67 33.99 72.88 93.75 11.00 20.0046.47 145.61 187.5 16.04 34.07 83.00 120.77 375 69.15 188.10 448.50392.44 750 133.95 304.30 524.02 526.58 1500 198.96 345.65 426.97 505.41VEGF-A Dose Containing N1-methyl-Pseudo-U/5mC 46.875 0.03 6.02 27.65100.42 93.75 12.37 46.38 121.23 167.56 187.5 104.55 365.71 1025.411056.91 375 605.89 1201.23 1653.63 1889.23 750 445.41 1036.45 1522.861954.81 1500 261.61 714.68 1053.12 1513.39

Example 39 In Vivo Studies of Factor IX

Human Factor IX mmRNA (Gen1; fully modified 5-methycytosine andpseudouridine) formulated in formulation buffer was delivered to micevia intramuscular injection. The results demonstrate that Factor IXprotein was elevated in serum as measured 13 hours after administration.

In this study, mice (N=5 for Factor IX, N=3 for Luciferase or Buffercontrols) were intramuscularly injected with 50 μl of the Factor IXmmRNA (mRNA sequence shown in SEQ ID NO: 10; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1), Luciferase (cDNAsequence for IVT shown in SEQ ID NO: 15; mRNA sequence shown in SEQ IDNO: 16, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) or theformulation buffer (F.Buffer) at 2×100 ug/mouse. The mice were bled at13 hours after the intramuscular injection to determine theconcentration of human the polypeptide in serum in pg/mL. The resultsrevealed that administration of Factor IX mmRNA resulted in levels of1600 pg/mL at 13 hours as compared to less than 100 pg/mL of Factor IXfor either Luciferase or buffer control administration.

Example 40 Multi-Site Administration: Intramuscular and Subcutaneous

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 6; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) modified as either Gen1 or Gen2 (5-methylcytosine (5mc) and apseudouridine (ψ) modification, G-CSF-Gen1; or N1-5-methylcytosine(N1-5mc) and a ψ modification, G-CSF-Gen2) and formulated in formulationbuffer were delivered to mice via intramuscular (IM) or subcutaneous(SC) injection. Injection of four doses or 2×50 ug (two sites) daily forthree days (24 hrs interval) was performed. The fourth dose wasadministered 6 hrs before blood collection and CBC analysis. Controlsincluded Luciferase (cDNA sequence for IVT shown in SEQ ID NO: 15; mRNAsequence shown in SEQ ID NO: 16, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer). The mice were bledat 72 hours after the first mRNA injection (6 hours after the last mRNAdose) to determine the effect of mRNA-encoded human G-CSF on theneutrophil count. The dosing regimen is shown in Table 63 as are theresulting neutrophil counts (thousands/uL). In Table 63, an asterisks(*)indicate statistical significance at p<0.05.

For intramuscular administration, the data reveal a four fold increasein neutrophil count above control at day 3 for the Gen1 G-CSF mRNA and atwo fold increase for the Gen2 G-CSF mmRNA. For subcutaneousadministration, the data reveal a two fold increase in neutrophil countabove control at day 3 for the Gen2 G-CSF mRNA.

These data demonstrate that both 5-methylcytidine/pseudouridine and5-methylcytidine/N1-methylpseudouridine-modified mRNA can bebiologically active, as evidenced by specific increases in bloodneutrophil counts.

TABLE 63 Dosing Regimen Dose Vol. Dosing Neutrophil Gr. Treatment RouteN = Dose (μg/mouse) (μl/mouse) Vehicle Thous/uL 1 G-CSF (Gen1) I.M 5 2 ×50 ug (four doses) 50 F. buffer 840* 2 G-CSF (Gen1) S.C 5 2 × 50 ug(four doses) 50 F. buffer 430 3 G-CSF (Gen2) I.M 5 2 × 50 ug (fourdoses) 50 F. buffer 746* 4 G-CSF (Gen2) S.C 5 2 × 50 ug (four doses) 50F. buffer 683 5 Luc (Gen1) I.M. 5 2 × 50 ug (four doses) 50 F. buffer201 6 Luc (Gen1) S.C. 5 2 × 50 ug (four doses) 50 F. buffer 307 7 Luc(Gen2) I.M 5 2 × 50 ug (four doses) 50 F. buffer 336 8 Luc (Gen2) S.C 52 × 50 ug (four doses) 50 F. buffer 357 9 F. Buffer I.M 4 0 (four doses)50 F. buffer 245 10 F. Buffer S.C. 4 0 (four doses) 50 F. buffer 509 11Untreated — 4 — 312

Example 41 Intravenous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 6; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) modified with 5-methylcytosine (5mc) and a pseudouridine (ψ)modification (Gen1); or having no modifications and formulated in 10%lipoplex (RNAiMax) were delivered to mice at a dose of 50 ug RNA and ina volume of 100 ul via intravenous (IV) injection at days 0, 2 and 4.Neutrophils were measured at days 1, 5 and 8. Controls includednon-specific mammalian RNA or the formulation buffer alone (F.Buffer).The mice were bled at days 1, 5 and 8 to determine the effect ofmRNA-encoded human G-CSF to increase neutrophil count. The dosingregimen is shown in Table 64 as are the resulting neutrophil counts(thousands/uL; K/uL).

For intravenous administration, the data reveal a four to five foldincrease in neutrophil count above control at day 5 with G-CSF modifiedmRNA but not with unmodified G-CSF mRNA or non-specific controls. Bloodcount returned to baseline four days after the final injection. No otherchanges in leukocyte populations were observed.

In Table 64, an asterisk(*) indicates statistical significance atp<0.001 compared to buffer.

These data demonstrate that lipoplex-formulated5-methylcytidine/pseudouridine-modified mRNA can be biologically active,when delivered through an I.V. route of administration as evidenced byspecific increases in blood neutrophil counts. No other cell subsetswere significantly altered. Unmodified G-CSF mRNA similarly administeredshowed no pharmacologic effect on neutrophil counts.

TABLE 64 Dosing Regimen Dose Vol. Dosing Neutrophil Gr. Treatment N =(μl/mouse) Vehicle K/uL 1 G-CSF (Gen1) Day 1 5 100 10% lipoplex 2.91 2G-CSF (Gen1) Day 5 5 100 10% lipoplex  5.32* 3 G-CSF (Gen1) Day 8 5 10010% lipoplex 2.06 4 G-CSF 5 100 10% lipoplex 1.88 (no modification) Day1 5 G-CSF 5 100 10% lipoplex 1.95 (no modification) Day 5 6 G-CSF 5 10010% lipoplex 2.09 (no modification) Day 8 7 RNA control Day 1 5 100 10%lipoplex 2.90 8 RNA control Day 5 5 100 10% lipoplex 1.68 9 RNA controlDay 8 4 100 10% lipoplex 1.72 10 F. Buffer Day 1 4 100 10% lipoplex 2.5111 F. Buffer Day 5 4 100 10% lipoplex 1.31 12 F. Buffer Day 8 4 100 10%lipoplex 1.92

Example 42 Saline Formulation: Intramuscular Administration

A. Protein Expression

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 6; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) and human EPO mmRNA (mRNA sequence shown in SEQ ID NO: 9; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1); G-CSF modified mRNA (modified with 5-methylcytosine (5mc) andpseudouridine (ψ)) and EPO modified mRNA (modified withN1-5-methylcytosine (N1-5mc) and ψ modification), were formulated informulation buffer (150 mM sodium chloride, 2 mM calcium chloride, 2 mMphosphate, 0.5 mM EDTA at a pH of 6.5) and delivered to mice viaintramuscular (IM) injection at a dose of 100 ug.

Controls included Luciferase (cDNA sequence for IVT, SEQ ID NO: 15; mRNAsequence shown in SEQ ID NO: 16, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer). The mice were bledat 13 hours after the injection to determine the concentration of thehuman polypeptide in serum in pg/mL (G-CSF groups measured human G-CSFin mouse serum and EPO groups measured human EPO in mouse serum). Thedata are shown in Table 65.

TABLE 65 Dosing Regimen Average Dose Dosing Protein Product GroupTreatment N = Vol. (μl/mouse) Vehicle Pg/mL, serum G-CSF G-CSF 5 50Saline 19.8 G-CSF Luciferase 5 50 Saline 0.5 G-CSF F. buffer 5 50 F.buffer 0.5 EPO EPO 5 50 Saline 191.5 EPO Luciferase 5 50 Saline 15.0 EPOF. buffer F. buffer 4.8

B. Dose Response

Human EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) were formulatedin formulation buffer and delivered to mice via intramuscular (IM)injection.

Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 16,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine and pseudouridine) orthe formulation buffer (F.Buffer). The mice were bled at 13 hours afterthe injection to determine the concentration of the human polypeptide inserum in pg/mL.

The dose and expression are shown in Table 66.

TABLE 66 Dosing Regimen and Expression Average Protein Dose Product Vol.pg/mL, Treatment (μl/mouse) serum EPO 100 96.2 EPO 50 63.5 EPO 25 18.7EPO 10 25.9 EPO 1 2.6 Luciferase 100 0.0 F. buffer 100 1.0

Example 43 Multi-Dose/Multi-Administration

Studies utilizing multiple intramuscular injection sites at one timepoint were designed and performed.

The design of a single multi-dose experiment involved using humanerythropoietin (EPO) mmRNA (mRNA sequence shown in SEQ ID NO: 9; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) or G-CSF (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)administered in formulation buffer. The dosing vehicle (F. buffer) wasused as a control. The EPO and G-CSF mmRNA were modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site.

Animals (n=5), Sprague-Dawley rats, were injected IM (intramuscular) forthe single unit dose of 100 ug (delivered to one thigh). Formulti-dosing 6 doses of 100 ug (delivered to two thighs) were used forboth EPO and G-CSF mmRNA. Control dosing involved use of buffer at asingle dose. Human EPO blood levels were evaluated 13 hrs postinjection.

Human EPO protein was measured in rat serum 13 h post I.M. Five groupsof rats were treated and evaluated. The results are shown in Table 67.

TABLE 67 Multi-dose study Avg. pg/mL Dose of Total human Group TreatmentmmRNA Dose EPO 1 Human EPO 1 × 100 ug 100 ug 143 mmRNA 2 Human EPO 6 ×100 ug 600 ug 256 mmRNA 3 G-CSF mmRNA 1 × 100 ug 100 ug 43 4 G-CSF mmRNA6 × 100 ug 600 ug 58 5 Buffer Alone — — 20

Example 44 Signal Sequence Exchange Study

Several variants of mmRNAs encoding human Granulocyte colony stimulatingfactor (G-CSF) (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) weresynthesized using modified nucleotides pseudouridine and5-methylcytosine (pseudo-U/5mC). These variants included the G-CSFconstructs encoding either the wild-type N terminal secretory signalpeptide sequence (MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 21), nosecretory signal peptide sequence, or secretory signal peptide sequencestaken from other mRNAs. These included sequences where the wild typeGCSF signal peptide sequence was replaced with the signal peptidesequence of either: human α-1-anti trypsin (AAT)(MMPSSVSWGILLLAGLCCLVPVSLA; SEQ ID NO: 22), human Factor IX (FIX)(MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR; SEQ ID NO: 23), humanProlactin (Prolac) (MKGSLLLLLVSNLLLCQSVAP; SEQ ID NO: 24), or humanAlbumin (Alb) (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 25).

250 ng of modified mRNA encoding each G-CSF variant was transfected intoHEK293A (293A in the table), mouse myoblast (MM in the table) (C2C12,CRL-1772, ATCC) and rat myoblast (RM in the table) (L6 line, CRL-1458,ATCC) cell lines in a 24 well plate using 1 ul of Lipofectamine 2000(Life Technologies), each well containing 300,000 cells. Thesupernatants were harvested after 24 hrs and the secreted G-CSF proteinwas analyzed by ELISA using the Human G-CSF ELISA kit (LifeTechnologies). The data shown in Table 68 reveal that cells transfectedwith G-CSF mmRNA encoding the Albumin signal peptide secrete at least 12fold more G-CSF protein than its wild type counterpart.

TABLE 68 Signal Peptide Exchange 293A MM RM Signal peptides (pg/ml)(pg/ml) (pg/ml) G-CSF Natural 9650 3450 6050 α-1-anti trypsin 9950 50008475 Factor IX 11675 6175 11675 Prolactin 7875 1525 9800 Albumin 12205081050 173300 No Signal peptide 0 0 0

Example 45 Cytokine Study: PBMC

PBMC Isolation and Culture:

50 mL of human blood from two donors was received from Research BloodComponents (lots KP30928 and KP30931) in sodium heparin tubes. For eachdonor, the blood was pooled and diluted to 70 mL with DPBS (SAFCBioscience 59331C, lot 071M8408) and split evenly between two 50 mLconical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot10074400) was gently dispensed below the blood layer. The tubes werecentrifuged at 2000 rpm for 30 minutes with low acceleration andbraking. The tubes were removed and the buffy coat PBMC layers weregently transferred to a fresh 50 mL conical and washed with DPBS. Thetubes were centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended andwashed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10minutes. This wash step was repeated, and the PBMC pellets wereresuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) andcounted. The cell suspensions were adjusted to a concentration of3.0×10̂6 cells/mL live cells.

These cells were then plated on five 96 well tissue culture treatedround bottom plates (Costar 3799) per donor at 50 uL per well. Within 30minutes, transfection mixtures were added to each well at a volume of 50uL per well. After 4 hours post transfection, the media was supplementedwith 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368)

Transfection Preparation:

mmRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO: 6; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) (containing either (1) natural NTPs, (2) 100% substitution with5-methyl cytidine and pseudouridine, or (3) 100% substitution with5-methyl cytidine and N1-methyl pseudouridine; mmRNA encoding luciferase(IVT cDNA sequence shown in SEQ ID NO: 15; mRNA sequence shown in SEQ IDNO: 16, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) (containingeither (1) natural NTPs or (2) 100% substitution with 5-methyl cytidineand pseudouridine) and TLR agonist R848 (Invivogen tlrl-r848) werediluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.

Separately, 432 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot1070962) was diluted with 13.1 mL Optimem I. In a 96 well plate ninealiquots of 135 uL of each mmRNA, positive control (R-848) or negativecontrol (Optimem I) was added to 135 uL of the diluted Lipofectamine2000. The plate containing the material to be transfected was incubatedfor 20 minutes. The transfection mixtures were then transferred to eachof the human PBMC plates at 50 uL per well. The plates were thenincubated at 37 C. At 2, 4, 8, 20, and 44 hours each plate was removedfrom the incubator, and the supernatants were frozen.

After the last plate was removed, the supernatants were assayed using ahuman G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit(Thermo Scientific 41105-2). Each condition was done in duplicate.

Results:

The ability of unmodified and modified mRNA (mmRNAs) to produce theencoded protein was assessed (G-CSF production) over time as was theability of the mRNA to trigger innate immune recognition as measured byinterferon-alpha production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102).

Results were interpolated against the standard curve of each ELISA plateusing a four parameter logistic curve fit. Shown in Tables 69 and 70 arethe average from 2 separate PBMC donors of the G-CSF and IFN-alphaproduction over time as measured by specific ELISA.

In the G-CSF ELISA, background signal from the Lipofectamine 2000untreated condition was subtracted at each timepoint. The datademonstrated specific production of human G-CSF protein by humanperipheral blood mononuclear is seen with G-CSF mRNA containing naturalNTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or100% substitution with 5-methyl cytidine and N1-methyl pseudouridine.Production of G-CSF was significantly increased through the use ofmodified mRNA relative to unmodified mRNA, with the 5-methyl cytidineand N1-methyl pseudouridine containing G-CSF mmRNA showing the highestlevel of G-CSF production. With regards to innate immune recognition,unmodified mRNA resulted in substantial IFN-alpha production, while themodified mRNA largely prevented interferon-alpha production. G-CSF mRNAfully modified with 5-methyl cytidine and N1-methylpseudouridine did notsignificantly increase cytokines whereas G-CSF mRNA fully modified with5-methyl cytidine and pseudouridine induced IFN-alpha, TNF-alpha andIP10. Many other cytokines were not affected by either modification.

TABLE 69 G-CSF Signal G-CSF signal - 2 Donor Average pg/mL 2 Hr 4 Hr 8Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine) 120.3 136.8 421.0 346.1 431.8G-CSF (5mC/N1-methyl 256.3 273.7 919.3 1603.3 1843.3 pseudouridine)GCSF(Natural-no modification) 63.5 92.6 129.6 258.3 242.4 Luciferase(5mC/pseudouridine) 4.5 153.7 33.0 186.5 58.0

TABLE 70 IFN-alpha signal IFN-alpha signal - 2 donor average pg/mL 2 Hr4 Hr 8 Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine) 21.1 2.9 3.7 22.7 4.3G-CSF (5mC/N1-methyl 0.5 0.4 3.0 2.3 2.1 pseudouridine) G-CSF(Natural)0.0 2.1 23.3 74.9 119.7 Luciferase (5mC/pseudouridine) 0.4 0.4 4.7 1.02.4 R-848 39.1 151.3 278.4 362.2 208.1 Lpf. 2000 control 0.8 17.2 16.50.7 3.1

Example 46 Chemical Modification Ranges of Modified mRNA

Modified nucleotides such as, but not limited to, the chemicalmodifications 5-methylcytosine and pseudouridine have been shown tolower the innate immune response and increase expression of RNA inmammalian cells. Surprisingly, and not previously known, the effectsmanifested by the chemical modifications can be titrated when the amountof chemical modification is less than 100%. Previously, it was believedthat full modification was necessary and sufficient to elicit thebeneficial effects of the chemical modifications and that less than 100%modification of an mRNA had little effect. However, it has now beenshown that the benefits of chemical modification can be derived usingless than complete modification and that the effects are target,concentration and modification dependent.

A. Modified RNA Transfected in PBMC

960 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.8 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, D3). The G-CSF mRNA (SEQID NO: 6; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) was completely modified with 5mC and pseudoU(100% modification), not modified with 5mC and pseudoU (0% modification)or was partially modified with 5mC and pseudoU so the mRNA would contain50% modification, 25% modification, 10% modification, %5 modification,1% modification or 0.1% modification. A control sample of Luciferase(mRNA sequence shown in SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified 5meC andpseudoU) was also analyzed for G-CSF expression. For TNF-alpha andIFN-alpha control samples of Lipofectamine-2000, LPS, R-848, Luciferase(mRNA sequence shown in SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified 5mC andpseudo), and P(I)P(C) were also analyzed. The supernatant was harvestedand run by ELISA 22 hours after transfection to determine the proteinexpression. The expression of G-CSF is shown in Table 71 and theexpression of IFN-alpha and TNF-alpha is shown in Table 72. Theexpression of IFN-alpha and TNF-alpha may be a secondary effect from thetransfection of the G-CSF mRNA. Tables and shows that the amount ofchemical modification of G-CSF, IFN-alpha and TNF-alpha is titratablewhen the mRNA is not fully modified and the titratable trend is not thesame for each target.

TABLE 71 G-CSF Expression G-CSF Expression (pg/ml) D1 D2 D3 100%modification 270.3 151.6 162.2  50% modification 45.6 19.8 26.3  25%modification 23.6 10.8 8.9  10% modification 39.4 12.9 12.9  5%modification 70.9 26.8 26.3  1% modification 70.3 26.9 66.9  0.1%modification 67.5 25.2 28.7 Luciferase 14.5 3.1 10.0

TABLE 72 IFN-alpha and TNF-alpha Expression IFN-alpha TNF-alphaExpression Expression (pg/ml) (pg/ml) D1 D2 D3 D1 D2 D3 100%modification 76.8 6.8 15.1 5.6 1.4 21.4  50% modification 22.0 5.5 257.34.7 1.7 12.1  25% modification 64.1 14.9 549.7 3.9 0.7 10.1  10%modification 150.2 18.8 787.8 6.6 0.9 13.4  5% modification 143.9 41.31009.6 2.5 1.8 12.0  1% modification 189.1 40.5 375.2 9.1 1.2 25.7  0.1%modification 261.2 37.8 392.8 9.0 2. 13.7  0% modification 230.3 45.1558.3 10.9 1.4 10.9 LF 200 0 0 1.5 45.8 2.8 53.6 LPS 0 0 1.0 114.5 70.0227.0 R-848 39.5 11.9 183.5 389.3 256.6 410.6 Luciferase 9.1 0 3.9 4.52.7 13.6 P(I)P(C) 1498.1 216.8 238.8 61.2 4.4 69.1

B. Modified RNA Transfected in HEK293

Human embryonic kidney epithelial (HEK293) cells were seeded on 96-wellplates at a density of 30,000 cells per well in 100 ul cell culturemedium. 250 ng of modified G-CSF mRNA (SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)formulated with RNAiMAX™ (Invitrogen, Carlsbad, Calif.) was added to awell. The G-CSF was completely modified with 5mC and pseudoU (100%modification), not modified with 5mC and pseudoU (0% modification) orwas partially modified with 5mC and pseudoU so the mRNA would contain75% modification, 50% modification or 25% modification. Control samples(AK 5/2, mCherry (SEQ ID NO: 7; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified 5mC andpseudoU) and untreated) were also analyzed. The half-life of G-CSF mRNAfully modified with 5-methylcytosine and pseudouridine is approximately8-10 hours. The supernatants were harvested after 16 hours and thesecreted G-CSF protein was analyzed by ELISA. Table 73 shows that theamount of chemical modification of G-CSF is titratable when the mRNA isnot fully modified.

TABLE 73 G-CSF Expression G-CSF Expression (ng/ml) 100% modification 118.4 75% modification 101.9 50% modification 105.7 25% modification231.1  0% modification 270.9 AK 5/2 166.8 mCherry 0 Untreated 0

Example 47 In Vivo Delivery of Modified mRNA (mmRNA)

Modified RNA was delivered to C57/BL6 mice intramuscularly,subcutaneously, or intravenously to evaluate the bio-distribution ofmodified RNA using luciferase. A formulation buffer used with alldelivery methods contained 150 mM sodium chloride, 2 mM calciumchloride, 2 mM Na⁺-phosphate which included 1.4 mM monobasic sodiumphosphate and 0.6 mM of dibasic sodium phosphate, and 0.5 mMethylenediaminetetraacetic acid (EDTA) was adjusted using sodiumhydroxide to reach a final pH of 6.5 before being filtered andsterilized. A IX concentration was used as the delivery buffer. Tocreate the lipoplexed solution delivered to the mice, in one vial 50 μgof RNA was equilibrated for 10 minutes at room temperature in thedelivery buffer and in a second vial 10 μl RNAiMAX™ was equilibrated for10 minutes at room temperature in the delivery buffer. Afterequilibrium, the vials were combined and delivery buffer was added toreach a final volume of 100 μl which was then incubated for 20 minutesat room temperature. Luciferin was administered by intraperitonealinjection (IP) at 150 mg/kg to each mouse prior to imaging during theplateau phase of the luciferin exposure curve which was between 15 and30 minutes. To create luciferin, 1 g of D-luciferin potassium or sodiumsalt was dissolved in 66.6 ml of distilled phosphate buffer solution(DPBS), not containing Mg2+ or Ca2+, to make a 15 mg/ml solution. Thesolution was gently mixed and passed through a 0.2 μm syringe filter,before being purged with nitrogen, aliquoted and frozen at −80° C. whilebeing protected from light as much as possible. The solution was thawedusing a waterbath if luciferin was not dissolved, gently mixed and kepton ice on the day of dosing.

Whole body images were taken of each mouse 2, 8 and 24 hours afterdosing. Tissue images and serum was collected from each mouse 24 hoursafter dosing. Mice administered doses intravenously had their liver,spleen, kidneys, lungs, heart, peri-renal adipose tissue and thymusimaged. Mice administered doses intramuscularly or subcutaneously hadtheir liver, spleen, kidneys, lungs, peri-renal adipose tissue, andmuscle at the injection site. From the whole body images thebioluminescence was measured in photon per second for each route ofadministration and dosing regimen.

A. Intramuscular Administration

Mice were intramuscularly (I.M.) administered either modified luciferasemRNA fully modified with 5-methylcytosine and pseudouridine (Naked-Luc),lipoplexed modified luciferase mRNA fully modified with 5-methylcytosineand pseudouridine (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO:15; mRNA sequence shown in SEQ ID NO: 16, polyA tail of approximately160 nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site), lipoplexed modified granulocyte colony-stimulating factor(G-CSF) mRNA (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) (Lipoplex-Cytokine) orthe formation buffer at a single dose of 50 μg of modified RNA in aninjection volume of 50 μl for each formulation in the right hind limband a single dose of 5 μg of modified RNA in an injection volume of 50μl in the left hind limb. The bioluminescence average for the luciferaseexpression signals for each group at 2, 8 and 24 hours after dosing areshown in Table 74. The bioluminescence showed a positive signal at theinjection site of the 5 μg and 50 μg modified RNA formulationscontaining and not containing lipoplex.

TABLE 74 In vivo Biophotoic Imaging (I.M. Injection Route) DoseBioluminescence (photon/sec) Formulation (ug) 2 hours 8 hours 24 hoursNaked-Luc 5 224,000 683,000 927,000 Lipolplex-Luc 5 579,000 639,000186,000 Lipoplex-G-CSF 5 64,600 85,600 75,100 Formulation Buffer 5102,000 86,000 90,700 Naked-Luc 50 446,000 766,000 509,000 Lipolplex-Luc50 374,000 501,000 332,000 Lipoplex-G-CSF 50 49,400 74,800 74,200Formulation Buffer 50 59,300 69,200 63,600

B. Subcutaneous Administration

Mice were subcutaneously (S.C.) administered either modified luciferasemRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation bufferat a single dose of 50 μg of modified mRNA in an injection volume of 100μl for each formulation. The bioluminescence average for the luciferaseexpression signals for each group at 2, 8 and 24 hours after dosing areshown in Table 75. The bioluminescence showed a positive signal at theinjection site of the 50 μg modified mRNA formulations containing andnot containing lipoplex.

TABLE 75 In vivo Biophotoic Imaging (S.C. Injection Route)Bioluminescence (photon/sec) Formulation 2 hours 8 hours 25 hoursNaked-Luc 3,700,000 8,060,000 2,080,000 Lipolplex-Luc 3,960,0001,700,000 1,290,000 Lipoplex-G-CSF 123,000 121,000 117,000 FormulationBuffer 116,000 127,000 123,000

C. Intravenous Administration

Mice were intravenously (I.V.) administered either modified luciferasemRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation bufferat a single dose of 50 μg of modified mRNA in an injection volume of 100μl for each formulation. The bioluminescence average for the luciferaseexpression signal in the spleen from each group at 2 hours after dosingis shown in Table 76. The bioluminescence showed a positive signal inthe spleen of the 50 μg modified mRNA formulations containing lipoplex.

TABLE 76 In vivo Biophotoic Imaging (I.V. Injection Route)Bioluminescence (photon/sec) Formulation of the Spleen Naked-Luc 58,400Lipolplex-Luc 65,000 Lipoplex-G-CSF 57,100 Formulation Buffer 58,300

Example 48 Split Dose Studies

Studies utilizing multiple subcutaneous or intramuscular injection sitesat one time point were designed and performed to investigate ways toincrease mmRNA drug exposure and improve protein production. In additionto detection of the expressed protein product, an assessment of thephysiological function of proteins was also determined through analyzingsamples from the animal tested.

Surprisingly, it has been determined that split dosing of mmRNA producesgreater protein production and phenotypic responses than those producedby single unit dosing or multi-dosing schemes.

The design of a single unit dose, multi-dose and split dose experimentinvolved using human erythropoietin (EPO) modified mRNA (mRNA shown inSEQ ID NO: 9; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) administered in buffer alone. The dosing vehicle(F. buffer) consisted of 150 mM NaCl, 2 mM CaCl₂, 2 mM Nat phosphate(1.4 mM monobasic sodium phosphate; 0.6 mM dibasic sodium phosphate),and 0.5 mM EDTA, pH 6.5. The pH was adjusted using sodium hydroxide andthe final solution was filter sterilized. The mmRNA was modified with5meC at each cytosine and pseudouridine replacement at each uridinesite.

Animals (n=5) were injected IM (intramuscular) for the single unit doseof 100 ug. For multi-dosing, two schedules were used, 3 doses of 100 ugand 6 doses of 100 ug. For the split dosing scheme, two schedules wereused, 3 doses at 33.3 ug and 6 doses of 16.5 ug mmRNA. Control dosinginvolved use of buffer only at 6 doses. Control mmRNA involved the useof luciferase mmRNA (IVT cDNA sequence shown in SEQ ID NO: 15; mRNAsequence shown in SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified 5meC ateach cytosine and pseudouridine replacement at each uridine site) dosed6 times at 100 ug. Blood and muscle tissue were evaluated 13 hrs postinjection.

Human EPO protein was measured in mouse serum 13 h post I.M. single,multi- or split dosing of the EPO mmRNA in buffer. Seven groups of mice(n=5 mice per group) were treated and evaluated. The results are shownin Table 77.

TABLE 77 Split dose study Avg. Polypeptide pmol/mL per unit Dose Dose ofTotal human drug Splitting Group Treatment mmRNA Dose EPO (pmol/ug)Factor 1 Human EPO mmRNA  1 × 100 ug 100 ug 14.3 0.14 1 2 Human EPOmmRNA  3 × 100 ug 300 ug 82.5 0.28 2 3 Human EPO mmRNA  6 × 100 ug 600ug 273.0 0.46 3.3 4 Human EPO mmRNA 3 × 33.3 ug 100 ug 104.7 1.1 7.9 5Human EPO mmRNA 6 × 16.5 ug 100 ug 127.9 1.3 9.3 6 Luciferase mmRNA  6 ×100 ug 600 ug 0 — — 7 Buffer Alone — — 0 — —

The splitting factor is defined as the product per unit drug divided bythe single dose product per unit drug (PUD). For example for treatmentgroup 2 the value 0.28 or product (EPO) per unit drug (mmRNA) is dividedby the single dose product per unit drug of 0.14. The result is 2.Likewise, for treatment group 4, the value 1.1 or product (EPO) per unitdrug (mmRNA) is divided by the single dose product per unit drug of0.14. The result is 7.9. Consequently, the dose splitting factor (DSF)may be used as an indicator of the efficacy of a split dose regimen. Forany single administration of a total daily dose, the DSF should be equalto 1. Therefore any DSF greater than this value in a split dose regimenis an indication of increased efficacy.

To determine the dose response trends, impact of injection site andimpact of injection timing, studies are performed. In these studies,varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and values in betweenare used to determine dose response outcomes. Split dosing for a 100 ugtotal dose includes three or six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6ug, or values and total doses equal to administration of the total doseselected.

Injection sites are chosen from the limbs or any body surface presentingenough area suitable for injection. This may also include a selection ofinjection depth to target the dermis (Intradermal), epidermis(Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection anglewill vary based on targeted delivery site with injections targeting theintradermal site to be 10-15 degree angles from the plane of the surfaceof the skin, between 20-45 degrees from the plane of the surface of theskin for subcutaneous injections and angles of between 60-90 degrees forinjections substantially into the muscle.

Example 49 Quantification in Exosomes

The quantity and localization of the mmRNA of the present invention canbe determined by measuring the amounts (initial, timecourse, or residualbasis) in isolated exosomes. In this study, since the mmRNA aretypically codon-optimized and distinct in sequence from endogenous mRNA,the levels of mmRNA are quantitated as compared to endogenous levels ofnative or wild type mRNA by using the methods of Gibbings,PCT/IB2009/005878, the contents of which are incorporated herein byreference in their entirety.

In these studies, the method is performed by first isolating exosomes orvesicles preferably from a bodily fluid of a patient previously treatedwith a modified mmRNA of the invention, then measuring, in saidexosomes, the modified mmRNA levels by one of mRNA microarray, qRT-PCR,or other means for measuring RNA in the art including by suitableantibody or immunohistochemical methods.

Example 50 Modified mRNA Transfection

A. Reverse Transfection

For experiments performed in a 24-well collagen-coated tissue cultureplate, Keratinocytes are seeded at a cell density of 1×10⁵. Forexperiments performed in a 96-well collagen-coated tissue culture plate,Keratinocytes are seeded at a cell density of 0.5×10⁵. For each modifiedmRNA (mmRNA) to be transfected, modified mRNA: RNAIMAX™ is prepared asdescribed and mixed with the cells in the multi-well plate within aperiod of time, e.g., 6 hours, of cell seeding before cells had adheredto the tissue culture plate.

B. Forward Transfection

In a 24-well collagen-coated tissue culture plate, Keratinocytes areseeded at a cell density of 0.7×10⁵. For experiments performed in a96-well collagen-coated tissue culture plate, Keratinocytes are seededat a cell density of 0.3×10⁵. Keratinocytes are grown to a confluencyof >70% for over 24 hours. For each modified mRNA (mmRNA) to betransfected, modified mRNA: RNAIMAX™ is prepared as described andtransfected onto the cells in the multi-well plate over 24 hours aftercell seeding and adherence to the tissue culture plate.

C. Modified mRNA Translation Screen: G-CSF ELISA

Keratinocytes are grown in EPILIFE medium with Supplement S7 fromInvitrogen (Carlsbad, Calif.) at a confluence of >70%. One set ofkeratinocytes were reverse transfected with 300 ng of the chemicallymodified mRNA (mmRNA) complexed with RNAIMAX™ from Invitrogen. Anotherset of keratinocytes are forward transfected with 300 ng modified mRNAcomplexed with RNAIMAX™ from Invitrogen. The modified mRNA: RNAIMAX™complex is formed by first incubating the RNA with Supplement-freeEPILIFE® media in a 5× volumetric dilution for 10 minutes at roomtemperature.

In a second vial, RNAIMAX™ reagent was incubated with Supplement-freeEPILIFE® Media in 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial was then mixed with the RNAIMAX™ vial andincubated for 20-30 minutes at room temperature before being added tothe cells in a drop-wise fashion. Secreted human Granulocyte-ColonyStimulating Factor (G-CSF) concentration in the culture medium ismeasured at 18 hours post-transfection for each of the chemicallymodified mRNA in triplicate.

Secretion of Human G-CSF from transfected human keratinocytes isquantified using an ELISA kit from Invitrogen or R&D Systems(Minneapolis, Minn.) following the manufacturers recommendedinstructions.

D. Modified mRNA Dose and Duration: G-CSF ELISA

Keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70%. Keratinocytes are reversetransfected with either 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750ng, or 1500 ng modified mRNA complexed with the RNAIMAX™ from Invitrogen(Carlsbad, Calif.). The modified mRNA:RNAIMAX™ complex is formed asdescribed. Secreted human G-CSF concentration in the culture medium ismeasured at 0, 6, 12, 24, and 48 hours post-transfection for eachconcentration of each modified mRNA in triplicate. Secretion of humanG-CSF from transfected human keratinocytes is quantified using an ELISAkit from Invitrogen or R&D Systems following the manufacturersrecommended instructions.

Example 51 Detection of a Cellular Innate Immune Response to ModifiedmRNA Using an ELISA Assay

An enzyme-linked immunosorbent assay (ELISA) for Human Tumor NecrosisFactor-α (TNF-α), Human Interferon-β (IFN-β) and HumanGranulocyte-Colony Stimulating Factor (G-CSF) secreted from invitro-transfected Human Keratinocyte cells is tested for the detectionof a cellular innate immune response. Keratinocytes are grown inEPILIFE® medium with Human Keratinocyte Growth Supplement in the absenceof hydrocortisone from Invitrogen (Carlsbad, Calif.) at a confluenceof >70%. Secreted TNF-α keratinocytes are reverse transfected with 0 ng,93.75 ng, 1 87.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of thechemically modified mRNA (mmRNA) complexed with RNAIMAX™ from Invitrogenas described in triplicate. Secreted TNF-α in the culture medium ismeasured 24 hours post-transfection for each of the chemically modifiedmRNA using an ELISA kit from Invitrogen according to the manufacturerprotocols.

Secreted IFN-β in the same culture medium is measured 24 hourspost-transfection for each of the chemically modified mRNA using anELISA kit from Invitrogen according to the manufacturer protocols.Secreted human G-CSF concentration in the same culture medium ismeasured at 24 hours post-transfection for each of the chemicallymodified mRNA. Secretion of human G-CSF from transfected humankeratinocytes is quantified using an ELISA kit from Invitrogen or R&DSystems (Minneapolis, Minn.) following the manufacturers recommendedinstructions. These data indicate which modified mRNA (mmRNA) arecapable eliciting a reduced cellular innate immune response incomparison to natural and other chemically modified polynucleotides orreference compounds by measuring exemplary type 1 cytokines TNF-α andIFN-β.

Example 52 Human Granulocyte-Colony Stimulating Factor (G-CSF) ModifiedmRNA-Induced Cell Proliferation Assay

Human keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70% in a 24-well collagen-coatedTRANSWELL® (Coming, Lowell, Mass.) co-culture tissue culture plate.Keratinocytes are reverse transfected with 750 ng of the indicatedchemically modified mRNA (mmRNA) complexed with RNAIMAX from Invitrogenas described in triplicate. The modified mRNA:RNAIMAX complex is formedas described. Keratinocyte media is exchanged 6-8 hourspost-transfection. 42-hours post-transfection, the 24-well TRANSWELL®plate insert with a 0.4 μm-pore semi-permeable polyester membrane isplaced into the human GCSF modified mRNA-transfected keratinocytecontaining culture plate

Human myeloblast cells, Kasumi-1 cells or KG-1 (0.2×10⁵ cells), areseeded into the insert well and cell proliferation is quantified 42hours post-co-culture initiation using the CyQuant Direct CellProliferation Assay (Invitrogen, Carlsbad, Calif.) in a 100-120 μlvolume in a 96-well plate. Modified mRNA-encoding human G-CSF-inducedmyeloblast cell proliferation is expressed as a percent cellproliferation normalized to untransfected keratinocyte/myeloblastco-culture control wells. Secreted human G-CSF concentration in both thekeratinocyte and myeloblast insert co-culture wells is measured at 42hours post-co-culture initiation for each modified mRNA in duplicate.Secretion of human G-CSF is quantified using an ELISA kit fromInvitrogen following the manufacturer recommended instructions.

Transfected human G-CSF modified mRNA in human keratinocyte feeder cellsand untransfected human myeloblast cells are detected by RT-PCR. TotalRNA from sample cells is extracted and lysed using RNEASY® kit (Qiagen,Valencia, Calif.) according to the manufacturer instructions. Extractedtotal RNA is submitted to RT-PCR for specific amplification of modifiedmRNA-G-CSF using PROTOSCRIPT® M-MuLV Taq RT-PCR kit (New EnglandBioLabs, Ipswich, Mass.) according to the manufacturer instructions withhuman G-CSF-specific primers. RT-PCR products are visualized by 1.2%agarose gel electrophoresis.

Example 53 Buffer Formulation Studies

G-CSF modified mRNA (SEQ ID NO: 6; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified withN1-pseudouridine and 5-methylcytosine) or Factor IX modified mRNA (SEQID NO: 10; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with N1-pseudouridine and5-methylcytosine) in a buffer solution is administered intramuscularlyto rats in an injection volume of 50 μl (n=5) at a modified mRNA dose of200 ug per rat as described in Table 78. The modified mRNA islyophilized in water for 1-2 days. It is then reconstituted in thebuffers listed below to a target concentration of 6 mg/ml. Concentrationis determined by OD 260. Samples are diluted to 4 mg/ml in theappropriate buffer before dosing.

To precipitate the modified mRNA, 3M sodium acetate, pH 5.5 and pureethanol are added at 1/10^(th) the total volume and 4 times the totalvolume of modified mRNA, respectively. The material is placed at −80 Cfor a minimum of 1 hour. The material is then centrifuged for 30 minutesat 4000 rpm, 4 C. The supernatant is removed and the pellet iscentrifuged and washed 3× with 75% ethanol. Finally, the pellet isreconstituted with buffer to a target concentration of 6 mg/ml.Concentration is determined by OD 260. Samples are diluted to 4 mg/ml inthe appropriate buffer before dosing. All samples are prepared bylyophilization unless noted below.

TABLE 78 Buffer Dosing Groups Group Treatment Buffer Dose (ug/rat) 1G-CSF 0.9% Saline 200 Factor IX 0.9% Saline 200 2 G-CSF 0.9% Saline + 2mM Calcium 200 Factor IX 0.9% Saline + 2 mM Calcium 200 3 G-CSF LactatedRinger's 200 Factor IX Lactated Ringer's 200 4 G-CSF 5% Sucrose 200Factor IX 5% Sucrose 200 5 G-CSF 5% Sucrose + 2 mM Calcium 200 Factor IX5% Sucrose + 2 mM Calcium 200 6 G-CSF 5% Mannitol 200 Factor IX 5%Mannitol 200 7 G-CSF 5% Mannitol + 2 mM Calcium 200 Factor IX 5%Mannitol + 2 mM Calcium 200 8 G-CSF 0.9% saline (precipitation) 200Factor IX 0.9% saline (precipitation) 200

Serum samples are collected from the rats at various time intervals andanalyzed for G-CSF or Factor IX protein expression using G-CSF or FactorIX ELISA.

Example 54 Multi-Dose Study

Sprague-Dawley rats (n=8; 4 female, 4 male) are injected intravenouslyeight times (twice a week) over 28 days. The rats are injected with 0.5mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg of human G-CSF modifiedmRNA of luciferase modified mRNA formulated in a lipid nanoparticle, 0.5mg/kg of human G-CSF modified mRNA in saline, 0.2 mg/kg of the humanG-CSF protein Neupogen or non-translatable human G-CSF modified mRNAformulated in a lipid nanoparticle. Serum is collected duringpre-determined time intervals to evaluate G-CSF protein expression (8,24 and 72 hours after the first dose of the week), complete blood countand white blood count (24 and 72 hours after the first dose of the week)and clinical chemistry (24 and 72 hours after the first dose of theweek). The rats are sacrificed at day 29, 4 days after the final dosing,to determine the complete blood count, white blood count, clinicalchemistry, protein expression and to evaluate the effect on the majororgans by histopathology and necropsy. Further, an antibody assay isperformed on the rats on day 29.

Example 55 Luciferase LNP In Vivo Study

Luciferase modified mRNA (SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine was formulated as a lipidnanoparticle (LNP) using the syringe pump method. The LNP was formulatedat a 20:1 weight ratio of total lipid to modified mRNA with a finallipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-DMG). As shown in Table 79, theluciferase LNP formulation was characterized by particle size, zetapotential, and encapsulation.

TABLE 79 Luciferase Formulation Formulation NPA-098-1 Modified mRNALuciferase Mean size 135 nm PDI: 0.08 Zeta at pH 7.4 −0.6 mV Encaps. 91%(RiboGr)

As outlined in Table 80, the luciferase LNP formulation was administeredto Balb-C mice (n=3) intramuscularly, intravenously and subcutaneouslyand a luciferase modified RNA formulated in PBS was administered to miceintravenously.

TABLE 80 Luciferase Formulations Concen- Injection Amount of trationVolume modified Dose Formulation Vehicle Route (mg/ml) (ul) RNA (ug)(mg/kg) Luc-LNP PBS IV 0.20 50 10 0.50 Luc-LNP PBS IM 0.20 50 10 0.50Luc-LNP PBS SC 0.20 50 10 0.50 Luc-PBS PBS IV 0.20 50 10 0.50

The mice administered the luciferase LNP formulation intravenously andintramuscularly were imaged at 2, 8, 24, 48, 120 and 192 hours and themice administered the luciferase LNP formulation subcutaneously wereimaged at 2, 8, 24, 48 and 120 hours to determine the luciferaseexpression as shown in Table 81. In Table 81, “NT” means not tested.Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

TABLE 81 Luciferase Expression Route of Average Expression(photon/second) Formulation Administration 2 hours 8 hours 24 hours 48hours 120 hours 192 hours Luc-LNP IV 1.62E+08 3.00E+09 7.77E+08 4.98E+081.89E+08 6.08E+07 Luc-LNP IM 4.85E+07 4.92E+08 9.02E+07 3.17E+071.22E+07 2.38E+06 Luc-LNP SC 1.85E+07 9.79E+08 3.09E+08 4.94E+071.98E+06 NT Luc-PBS IV 3.61E+05 5.64E+05 3.19E+05 NT NT NT

One mouse administered the LNP formulation intravenously was sacrificedat 8 hours to determine the luciferase expression in the liver andspleen. Also, one mouse administered the LNP formulation intramuscularwas sacrificed at 8 hours to determine the luciferase expression of themuscle around the injection site and in the liver and spleen. As shownin Table 82, expression was seen in the both the liver and spleen afterintravenous and intramuscular administration and in the muscle aroundthe intramuscular injection site.

TABLE 82 Luciferase Expression in Tissue Expression (photon/second)Luciferase LNP: IV Administration Liver 7.984E+08 Spleen 3.951E+08Luciferase LNP: IM Administration Muscle around the 3.688E+07 injectionsite Liver 1.507E+08 Spleen 1.096E+07

Example 56 In Vitro PBMC Studies: Percent Modification

480 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(SEQ ID NO: 6; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) was completely modified with 5mC and pseudoU(100% modification), not modified with 5mC and pseudoU (0% modification)or was partially modified with 5mC and pseudoU so the mRNA would contain75% modification, 50% modification or 25% modification. A control sampleof Luciferase (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified 5meC and pseudoU) was also analyzed for G-CSF expression. ForTNF-alpha and IFN-alpha control samples of Lipofectamine-2000, LPS,R-848, Luciferase (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified 5mC and pseudo), and P(I)P(C) were also analyzed. Thesupernatant was harvested and run by ELISA 22 hours after transfectionto determine the protein expression. The expression of G-CSF is shown inTable 83 and the expression of IFN-alpha and TNF-alpha is shown in Table84. The expression of IFN-alpha and TNF-alpha may be a secondary effectfrom the transfection of the G-CSF mRNA. Tables 83 and 84 show that theamount of chemical modification of G-CSF, interferon alpha (IFN-alpha)and tumor necrosis factor-alpha (TNF-alpha) is titratable when the mRNAis not fully modified and the titratable trend is not the same for eachtarget.

As mentioned above, using PBMC as an in vitro assay system it ispossible to establish a correlation between translation (in this caseG-CSF protein production) and cytokine production (in this caseexemplified by IFN-alpha protein production). Better protein productionis correlated with lower induction of innate immune activation pathway,and the percentage modification of a chemistry can be judged favorablybased on this ratio (Table 85). As calculated from Tables 83 and 84 andshown in Table 85, full modification with 5-methylcytidine andpseudouridine shows a much better ratio of protein cytokine productionthan without any modification (natural G-CSF mRNA) (100-fold forIFN-alpha and 27-fold for TNF-alpha). Partial modification shows alinear relationship with increasingly less modification resulting in alower protein cytokine ratio.

TABLE 83 G-CSF Expression G-CSF Expression (pg/ml) D1 D2 D3 100%modification  1968.9 2595.6 2835.7 75% modification 566.7 631.4 659.550% modification 188.9 187.2 191.9 25% modification 139.3 126.9 102.0 0% modification 194.8 182.0 183.3 Luciferase 90.2 0.0 22.1

TABLE 84 IFN-alpha and TNF-alpha Expression IFN-alpha TNF-alphaExpression (pg/ml) Expression (pg/ml) D1 D2 D3 D1 D2 D3 100%modification 336.5 78.0 46.4 115.0 15.0 11.1  75% modification 339.6107.6 160.9 107.4 21.7 11.8  50% modification 478.9 261.1 389.7 49.624.1 10.4  25% modification 564.3 400.4 670.7 85.6 26.6 19.8  0%modification 1421.6 810.5 1260.5 154.6 96.8 45.9 LPS 0.0 0.6 0.0 0.012.6 4.3 R-848 0.5 3.0 14.1 655.2 989.9 420.4 P(I)P(C) 130.8 297.1 585.2765.8 2362.7 1874.4 Lipid only 1952.2 866.6 855.8 248.5 82.0 60.7

TABLE 85 PC Ratio and Effect of Percentage of Modification AverageAverage Average G-CSF/IFN- G-CSF/TNF- G-CSF IFN-a TNF-a alpha alpha %Modification (pg/ml) (pg/ml) (pg/ml) (PC ratio) (PC ratio) 100 2466 15347 16 52 75 619 202 47 3.1 13 50 189 376 28 0.5 6.8 25 122 545 44 0.22.8 0 186 1164 99 0.16 1.9

Example 57 Modified RNA Transfected in PBMC

500 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine

(pseudoU) or unmodified G-CSF mRNA was transfected with 0.4 uL ofLipofectamine 2000 into peripheral blood mononuclear cells (PBMC) fromthree normal blood donors (D1, D2, and D3). The G-CSF mRNA (SEQ ID NO:6; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was completely modified with 5mC and pseudoU (100%modification), not modified with 5mC and pseudoU (0% modification) orwas partially modified with 5mC and pseudoU so the mRNA would contain50% modification, 25% modification, 10% modification, %5 modification,1% modification or 0.1% modification. A control sample of mCherry (mRNAsequence shown in SEQ ID NO: 7; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified 5meC andpseudouridine) and G-CSF fully modified with 5-methylcytosine andpseudouridine (Control G-CSF) was also analyzed for G-CSF expression.For tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha(IFN-alpha) control samples of Lipofectamine-2000, LPS, R-848,Luciferase (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified 5mC and pseudo), and P(I)P(C) were also analyzed. Thesupernatant was harvested 6 hours and 18 hours after transfection andrun by ELISA to determine the protein expression. The expression ofG-CSF, IFN-alpha, and TNF-alpha for Donor 1 is shown in Table 86, Donor2 is shown in Table 87 and Donor 3 is shown in Table 88.

Full 100% modification with 5-methylcytidine and pseudouridine resultedin the most protein translation (G-CSF) and the least amount of cytokineproduced across all three human PBMC donors. Decreasing amounts ofmodification results in more cytokine production (IFN-alpha andTNF-alpha), thus further highlighting the importance of fullymodification to reduce cytokines and to improve protein translation (asevidenced here by G-CSF production).

TABLE 86 Donor 1 G-CSF (pg/mL) IFN-alpha (pg/mL) TNF-alpha (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 1815 2224 1 130 0  75% Mod 591 614 0 89 0 0  50% Mod 172 147 0 193 0 0  25% Mod 111 922 219 0 0  10% Mod 138 138 7 536 18 0  1% Mod 199 214 9 660 18 3  0.1%Mod 222 208 10 597 0 6  0% Mod 273 299 10 501 10 0 Control G-CSF 9571274 3 123 18633 1620 mCherry 0 0 0 10 0 0 Untreated N/A N/A 0 0 1 1

TABLE 87 Donor 2 G-CSF (pg/mL) IFN-alpha (pg/mL) TNF-alpha (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 2184 2432 0 70 11  75% Mod 935 958 3 130 0 0  50% Mod 192 253 2 625 7 23  25% Mod 153158 7 464 6 6  10% Mod 203 223 25 700 22 39  1% Mod 288 275 27 962 51 66 0.1% Mod 318 288 33 635 28 5  0% Mod 389 413 26 748 1 253 Control G-CSF1461 1634 1 59 481 814 mCherry 0 7 0 1 0 0 Untreated N/A N/A 1 0 0 0

TABLE 88 Donor 3 G-CSF (pg/mL) IFN-alpha (pg/mL) TNF-alpha (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 6086 7549 7658 11 11  75% Mod 2479 2378 23 752 4 35  50% Mod 667 774 24 896 22 18 25% Mod 480 541 57 1557 43 115  10% Mod 838 956 159 2755 144 123  1%Mod 1108 1197 235 3415 88 270  0.1% Mod 1338 1177 191 2873 37 363  0%Mod 1463 1666 215 3793 74 429 Control G-CSF 3272 3603 16 1557 731 9066mCherry 0 0 2 645 0 0 Untreated N/A N/A 1 1 0 8

Example 58 Innate Immune Response Study in BJ Fibroblasts

A. Single Transfection

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 6; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methylpseudouridine (Gen2) having Cap0, Cap1 or no cap wastransfected using Lipofectamine 2000 (Invitrogen, catalog #11668-019),following manufacturer's protocol. Control samples of poly I:C (PIC),Lipofectamine 2000 (Lipo), natural luciferase mRNA (mRNA sequence shownin SEQ ID NO: 16; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) and natural G-CSF mRNA were also transfected.The cells were harvested after 18 hours, the total RNA was isolated andDNASE® treated using the RNeasy micro kit (catalog #74004) following themanufacturer's protocol. 100 ng of total RNA was used for cDNA synthesisusing High Capacity cDNA Reverse Transcription kit (catalog #4368814)following the manufacturer's protocol. The cDNA was then analyzed forthe expression of innate immune response genes by quantitative real timePCR using SybrGreen in a Biorad CFX 384 instrument followingmanufacturer's protocol. Table 89 shows the expression level of innateimmune response transcripts relative to house-keeping gene HPRT(hypoxanthine phosphoribosytransferase) and is expressed asfold-induction relative to HPRT. In the table, the panel of standardmetrics includes: RIG-I is retinoic acid inducible gene 1, IL6 isinterleukin-6, OAS-1 is oligoadenylate synthetase 1, IFNb isinterferon-beta, AIM2 is absent in melanoma-2, IFIT-1 isinterferon-induced protein with tetratricopeptide repeats 1, PKR isprotein kinase R, TNFa is tumor necrosis factor alpha and IFNa isinterferon alpha.

TABLE 89 Innate Immune Response Transcript Levels Formulation RIG-I IL6OAS-1 IFNb AIM2 IFIT-1 PKR TNFa IFNa Natural 71.5 20.6 20.778 11.4040.251 151.218 16.001 0.526 0.067 Luciferase Natural G- 73.3 47.1 19.35913.615 0.264 142.011 11.667 1.185 0.153 CSF PIC 30.0 2.8 8.628 1.5230.100 71.914 10.326 0.264 0.063 G-CSF Gen1- 0.81 0.22 0.080 0.009 0.0082.220 1.592 0.090 0.027 UC G-CSF Gen1- 0.54 0.26 0.042 0.005 0.008 1.3141.568 0.088 0.038 Cap0 G-CSF Gen1- 0.58 0.30 0.035 0.007 0.006 1.5101.371 0.090 0.040 Cap1 G-CSF Gen2- 0.21 0.20 0.002 0.007 0.007 0.6030.969 0.129 0.005 UC G-CSF Gen2- 0.23 0.21 0.002 0.0014 0.007 0.6481.547 0.121 0.035 Cap0 G-CSF Gen2- 0.27 0.26 0.011 0.004 0.005 0.6781.557 0.099 0.037 Cap1 Lipo 0.27 0.53 0.001 0 0.007 0.954 1.536 0.1580.064

B. Repeat Transfection

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 6; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1) unmodified, fully modified with5-methylcytosine and pseudouridine (Gen1) or fully modified with5-methylcytosine and N1-methylpseudouridine (Gen2) was transfected dailyfor 5 days following manufacturer's protocol. Control samples ofLipofectamine 2000 (L2000) and mCherry mRNA (mRNA sequence shown in SEQID NO: 7; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine andpseudouridine) were also transfected daily for 5 days. The results areshown in Table 90.

Unmodified mRNA showed a cytokine response in interferon-beta (IFN-beta)and interleukin-6 (IL-6) after one day. mRNA modified with at leastpseudouridine showed a cytokine response after 2-3 days whereas mRNAmodified with 5-methylcytosine and N1-methylpseudouridine showed areduced response after 3-5 days.

TABLE 90 Cytokine Response Formulation Transfection IFN-beta (pg/ml)IL-6 (pg/ml) G-CSF unmodified 6 hours 0 3596 Day 1 1363 15207 Day 2 23812415 Day 3 225 5017 Day 4 363 4267 Day 5 225 3094 G-CSF Gen 1 6 hours 03396 Day 1 38 3870 Day 2 1125 16341 Day 3 100 25983 Day 4 75 18922 Day 5213 15928 G-CSF Gen 2 6 hours 0 3337 Day 1 0 3733 Day 2 150 974 Day 3213 4972 Day 4 1400 4122 Day 5 350 2906 mCherry 6 hours 0 3278 Day 1 2383893 Day 2 113 1833 Day 3 413 25539 Day 4 413 29233 Day 5 213 20178L2000 6 hours 0 3270 Day 1 13 3933 Day 2 388 567 Day 3 338 1517 Day 4475 1594 Day 5 263 1561

Example 59 In Vivo Detection of Innate Immune Response Study

Female BALB/C mice (n=5) were injected intramuscularly with G-CSF mRNA(GCSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence;) with a 5′ cap ofCap1, G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine(GCSF mRNA 5mc/pU), G-CSF mRNA fully modified with 5-methylcytosine andN1-methylpseudouridine with (GCSF mRNA 5mc/N1pU) or without a 5′ cap(GCSF mRNA 5mc/N1 pU no cap) or a control of either R848 or 5% sucroseas described in Table 91. Blood is collected at 8 hours after dosing andusing ELISA the protein levels of G-CSF and interferon-alpha (IFN-alpha)is determined by ELISA and are shown in Table 81.

As shown in Table 91, unmodified, 5mc/pU, and 5mc/N1pU modified G-CSFmRNA resulted in human G-CSF expression in mouse serum. The uncapped5mC/N1pU modified G-CSF mRNA showed no human G-CSF expression in serum,highlighting the importance of having a 5′ cap structure for proteintranslation.

As expected, no human G-CSF protein was expressed in the R848, 5%sucrose only, and untreated groups. Importantly, significant differenceswere seen in cytokine production as measured by mouse IFN-alpha in theserum. As expected, unmodified G-CSF mRNA demonstrated a robust cytokineresponse in vivo (greater than the R848 positive control). The 5mc/pUmodified G-CSF mRNA did show a low but detectable cytokine response invivo, while the 5mc/N1pU modified mRNA showed no detectable IFN-alpha inthe serum (and same as vehicle or untreated animals).

Also, the response of 5mc/N1pU modified mRNA was the same regardless ofwhether it was capped or not. These in vivo results reinforce theconclusion that 1) that unmodified mRNA produce a robust innate immuneresponse, 2) that this is reduced, but not abolished, through 100%incorporation of 5mc/pU modification, and 3) that incorporation of5mc/N1pU modifications results in no detectable cytokine response.

Lastly, given that these injections are in 5% sucrose (which has noeffect by itself), these result should accurately reflect theimmunostimulatory potential of these modifications.

From the data it is evident that N1pU modified molecules produce moreprotein while concomitantly having little or no effect on IFN-alphaexpression. It is also evident that capping is required for proteinproduction for this chemical modification. The Protein: Cytokine Ratioof 748 as compared to the PC Ratio for the unmodified mRNA (PC=9) meansthat this chemical modification is far superior as related to theeffects or biological implications associated with IFN-alpha.

TABLE 91 Human G-CSF and Mouse IFN-alpha in serum G-CSF IFN-alpha DoseDose protein expression PC Formulation Route (ug/mouse) (ul) (pg/ml)(pg/ml) Ratio GCSF mRNA unmod I.M. 200 50 605.6 67.01 9 GCSF mRNA 5mc/pUI.M. 200 50 356.5 8.87 40 GCSF mRNA5mc/N1pU I.M. 200 50 748.1 0 748 GCSFmRNA5mc/N1pU no cap I.M. 200 50 6.5 0 6.5 R848 I.M.  75 50 3.4 40.97 .085% sucrose I.M. — 50 0 1.49 0 Untreated I.M. — — 0 0 0

Example 60 In Vivo Delivery of Modified RNA

Protein production of modified mRNA was evaluated by delivering modifiedG-CSF mRNA or modified Factor IX mRNA to female Sprague Dawley rats(n=6). Rats were injected with 400 ug in 100 ul of G-CSF mRNA (mRNAsequence shown in SEQ ID NO: 6; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (G-CSF Gen1), G-CSF mRNA fullymodified with 5-methylcytosine and N1-methylpseudouridine (G-CSF Gen2)or Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 10; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (Factor IX Gen1)reconstituted from the lyophilized form in 5% sucrose. Blood wascollected 8 hours after injection and the G-CSF protein level in serumwas measured by ELISA. Table 92 shows the G-CSF protein levels in serumafter 8 hours.

These results demonstrate that both G-CSF Gen 1 and G-CSF Gen 2 modifiedmRNA can produce human G-CSF protein in a rat following a singleintramuscular injection, and that human G-CSF protein production isimproved when using Gen 2 chemistry over Gen 1 chemistry.

TABLE 92 G-CSF Protein in Rat Serum (I.M. Injection Route) FormulationG-CSF protein (pg/ml) G-CSF Gen1 19.37 G-CSF Gen2 64.72 Factor IX Gen 12.25

Example 61 Stability of Modified RNA

Stability experiments were conducted to obtain a better understanding ofstorage conditions to retain the integrity of modified RNA. UnmodifiedG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 6; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine and G-CSF mRNA fullymodified with 5-methylcytosine and pseudouridine lipoplexed with 0.75%by volume of RNAIMAX™ was stored at 50° C., 40° C., 37° C., 25° C., 4°C. or −20° C. After the mRNA had been stored for 0 hours, 2 hours, 6hours, 24 hours, 48 hours, 5 days and 14 days, the mRNA was analyzed bygel electrophoresis using a Bio-Rad EXPERION™ system. The modified,unmodified and lipoplexed G-CSF mRNA was also stored in RNASTABLE®(Biomatrica, Inc. San Diego, Calif.) at 40° C. or water at −80° C. or40° C. for 35 days before being analyzed by gel electrophoresis.

All mRNA samples without stabilizer were stable after 2 weeks afterstorage at 4° C. or −20° C. Modified G-CSF mRNA, with or withoutlipoplex, was more stable than unmodified G-CSF when stored at 25° C.(stable out to 5 days versus 48 hours), 37° C. (stable out to 24 hoursversus 6 hours) and 50° C. (stable out to 6 hours versus 2 hours).Unmodified G-CSF mRNA, modified G-CSF mRNA with or without lipoplextolerated 12 freeze/thaw cycles.

mRNA samples stored in stabilizer at 40° C. showed similar stability tothe mRNA samples stored in water at −80° C. after 35 days whereas themRNA stored in water at 40° C. showed heavy degradation after 18 days.

mRNA samples stored at 4° C., 25° C. and 37° C. were stored in 1×TEbuffer or the formulation buffer (150 mM sodium chloride, 2 mM calciumchloride, 2 mM phosphate, 0.5 mM EDTA at a pH of 6.5). The mRNA storedat 4° C. was stable to at least 60 days in both the TE and formulationbuffer. At 25° C. the mRNA in formulation buffer was stable out to 14days and the TE buffer was stable out to at least 6 days. Storage ofmRNA in the formulation buffer at 37° C. was stable to 6 days comparedto the TE buffer which was stable only until 4 days.

Example 62 Effects of Chemical Modifications on Expression of FormulatedmRNA

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and 2′Fluorouridine is formulated in saline orDLin-MC3-DMA and administered intravenously, intramuscularly orsubcutaneously to rodents at a dose of 0.5 mg/kg, 0.05 mg/kg, 0.005mg/kg and/or 0.0005 mg/kg. Luciferase mRNA (SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine is formulated inDLin-MC3-DMA and administered intramuscularly or subcutaneously torodents at a dose of 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg and/or 0.0005mg/kg. The DLin-MC3-DMA formulations are analyzed prior toadministration to determine the mean size and zeta potential. Therodents are imaged at 2 hours, 8 hours, 24 hours, 72 hours, 96 hours,144 hours and 168 hours after dosing and the bioluminescence is measuredin photon per second for each route of administration and formulation.

Example 63 Expression of PLGA Formulated mRNA

A. Synthesis and Characterization of Luciferase PLGA Microspheres

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl pseudouridine, modifiedwith 25% of uridine replaced with 2-thiouridine and 25% of cytosinereplaced with 5-methylcytosine, fully modified with N1-methylpseudouridine, or fully modified with pseudouridine was reconstituted in1×TE buffer and then formulated in PLGA microspheres. PLGA microsphereswere synthesized using the water/oil/water double emulsification methodsknown in the art using PLGA-ester cap (Lactel, Cat# B6010-2, inherentviscosity 0.55-0.75, 50:50 LA:GA), polyvinylalcohol (PVA) (Sigma,Cat#348406-25G, MW 13-23 k) dichloromethane and water. Briefly, 0.4 mlof mRNA in TE buffer at 4 mg/ml (W1) was added to 2 ml of PLGA dissolvedin dichloromethane (DCM) (O1) at a concentration of 200 mg/ml of PLGA.The W1/O1 emulsion was homogenized (IKA Ultra-Turrax Homogenizer, T18)for 30 seconds at speed 5 (˜19,000 rpm). The W1/O1 emulsion was thenadded to 250 ml 1% PVA (W2) and homogenized for 1 minute at speed 5(˜19,000 rpm). Formulations were left to stir for 3 hours, then passedthrough a 100 μm nylon mesh strainer (Fisherbrand Cell Strainer, Cat#22-363-549) to remove larger aggregates, and finally washed bycentrifugation (10 min, 9,250 rpm, 4° C.). The supernatant was discardedand the PLGA pellets were resuspended in 5-10 ml of water, which wasrepeated 2×. After washing and resuspension with water, 100-200 μl of aPLGA microspheres sample was used to measure particle size of theformulations by laser diffraction (Malvern Mastersizer2000). The washedformulations were frozen in liquid nitrogen and then lyophilized for 2-3days.

After lyophilization, ˜10 mg of PLGA MS were weighed out in 2 mleppendorf tubes and deformulated by adding 1 ml of DCM and letting thesamples shake for 2-6 hrs. The mRNA was extracted from the deformulatedPLGA micropsheres by adding 0.5 ml of water and shaking the sampleovernight. Unformulated luciferase mRNA in TE buffer (unformulatedcontrol) was spiked into DCM and went through the deformulation process(deformulation control) to be used as controls in the transfectionassay. The encapsulation efficiency, weight percent loading and particlesize are shown in Table 93. Encapsulation efficiency was calculated asmg of mRNA from deformulation of PLGA microspheres divided by theinitial amount of mRNA added to the formulation. Weight percent loadingin the formulation was calculated as mg of mRNA from deformulation ofPLGA microspheres divided by the initial amount of PLGA added to theformulation.

TABLE 93 PLGA Characteristics Theoretical Particle mRNA Actual mRNA SizeEncapsulation Loading (wt Loading (wt (D50, Chemical ModificationsSample ID Efficiency (%) %) %) um) Fully modified with 5- 43-66A 45.80.4 0.18 33.4 methylcytosine and N1- 43-66B 29.6 0.12 27.7 methylpseudouridine 43-66C 25.5 0.10 27.1 25% of uridine replaced 43-67A 34.60.4 0.14 29.9 with 2-thiouridine and 43-67B 22.8 0.09 30.2 25% ofcytosine replaced 43-67C 23.9 0.10 25.1 with 5-methylcytosine Fullymodified with N1- 43-69A 55.8 0.4 0.22 40.5 methyl pseudouridine 43-69B31.2 0.12 41.1 43-69C 24.9 0.10 46.1 Fully modified with 43-68-1 49.30.4 0.20 34.8 pseudouridine 43-68-2 37.4 0.15 35.9 43-68-3 45.0 0.1836.5

B. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 83 ng ofthe deformulated luciferase mRNA PLGA microsphere samples, deformulatedluciferase mRNA control (Deform control), or unformulated luciferasemRNA control (Unfomul control) was diluted in a 10 ul final volume ofOPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as a transfectionreagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM.After 5 min of incubation at room temperature, both solutions werecombined and incubated an additional 15 min at room temperature. Then 20ul of the combined solution was added to 100 ul of cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After a 18 to 22 hour incubation, cells expressing luciferase were lysedwith 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.).

Cells were harvested and the bioluminescence (in relative light units,RLU) for each sample is shown in Table 94. Transfection of these samplesconfirmed that the varied chemistries of luciferase mRNA is still ableto express luciferase protein after PLGA microsphere formulation.

TABLE 94 Chemical Modifications Chemical Biolum. Modifications Sample ID(RLU) Fully modified with Deform contol 164266.5 5-methylcytosineUnformul control 113714 and N1-methyl 43-66A 25174 pseudouridine 43-66B25359 43-66C 20060 25% of uridine Deform contol 90816.5 replaced with 2-Unformul control 129806 thiouridine and 25% 43-67A 38329.5 of cytosinereplaced 43-67B 8471.5 with 5- 43-67C 10991.5 methylcytosine Fullymodified with Deform contol 928093.5 N1-methyl Unformul control1512273.5 pseudouridine 43-69A 1240299.5 43-69B 748667.5 43-69C 1193314Fully modified with Deform contol 154168 pseudouridine Unformul control151581 43-68-1 120974.5 43-68-2 107669 43-68-3 97226

Example 64 In Vitro Studies of Factor IX A. Serum-Free Media

Human Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 10; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) was transfectedin serum-free media. The cell culture supernatant was collected andsubjected to trypsin digestion before undergoing 2-dimensional HPLCseparation of the peptides. Matrix-assisted laser desorption/ionizationwas used to detect the peptides. 8 peptides were detected and 7 of thedetected peptides are unique to Factor IX. These results indicate thatthe mRNA transfected in the serum-free media was able to expressfull-length Factor IX protein.

B. Human Embryonic Kidney (HEK) 293A Cells

250 ng of codon optimized Human Factor IX mRNA (mRNA sequence shown inSEQ ID NO: 10; fully modified with 5-methylcytosine and pseudouridine;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was transfected into HEK 293A cells (150,000 cells/well)using Lipofectamine 2000 in DMEM in presence of 10% FBS. Thetransfection complexes were removed 3 hours after transfection. Cellswere harvested at 3, 6, 9, 12, 24, 48 and 72 hours after transfection.Total RNA was isolated and used for cDNA synthesis. The cDNA wassubjected to analysis by quantitative Real-Time PCR using codonoptimized Factor IX specific primer set. Human hypoxanthinephosphoribosyltransfersase 1 (HPRT) level was used for normalization.The data is plotted as a percent of detectable mRNA considering the mRNAlevel as 100% at the 3 hour time point. The half-life of Factor IXmodified mRNA fully modified with 5-methylcytosine and pseudouridine inhuman embryonic kidney 293 (HEK293) cells is about 8-10 hours.

Example 65 Saline Formulation: Subcutaneous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 6; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) and humanEPO modified mRNA (mRNA sequence shown in SEQ ID NO: 9; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine), were formulated insaline and delivered to mice via intramuscular (IM) injection at a doseof 100 ug.

Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 16;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine)) orthe formulation buffer (F.Buffer). The mice were bled at 13 hours afterthe injection to determine the concentration of the human polypeptide inserum in pg/mL. (G-CSF groups measured human G-CSF in mouse serum andEPO groups measured human EPO in mouse serum). The data are shown inTable 95.

mRNA degrades rapidly in serum in the absence of formulation suggestingthe best method to deliver mRNA to last longer in the system is byformulating the mRNA. As shown in Table 95, mRNA can be deliveredsubcutaneously using only a buffer formulation.

TABLE 95 Dosing Regimen Average Dose Protein Vol. Product (μl/ Dosingpg/mL, Group Treatment mouse) Vehicle serum G-CSF G-CSF 100 F. buffer 45G-CSF Luciferase 100 F. buffer 0 G-CSF F. buffer 100 F. buffer 2.2 EPOEPO 100 F. buffer 72.03 EPO Luciferase 100 F. buffer 26.7 EPO F. buffer100 F. buffer 13.05

Example 66 Stability of Nanoparticle of Formulations

Formulations of DLin-KC2-DMA, Teta-5-Lap, DLin-DMA, DLin-K-DMA, C12-200,DLin-MC3-DMA at a lipid:mRNA ratio of 20:1 were evaluated for particlesize, polydispersity index and encapsulation efficiency for stability atroom temperature. Most nanoparticles are stable at room temperature forat least one month as shown in Tables 96 and 97.

TABLE 96 Particle Size and Polydispersity Index Formulation Time # Lipid0 hours 24 hours 48 hours 30 days NPA-003-4 DLin- 112 nm 110 nm 103 nm104 nm KC2- PDI: 0.05 PDI: 0.06 PDI: 0.09 PDI: 0.08 DMA NPA-006-2Teta-5- 95 nm 95 nm 95 nm 100 nm Lap PDI: 0.09 PDI: 012 PDI: 0.10 PDI:0.11 NPA-012-1 DLin-  90 nm  87 nm  89 nm  82 nm DMA PDI: 0.09 PDI: 0.07PDI: 0.08 PDI: 0.08 NPA-013-1 DLin-K-  92 nm  91 nm  96 nm  91 nm DMAPDI: 0.07 PDI: 0.06 PDI: 0.05 PDI: 0.06 NPA-014-1 C12-200  99 nm  98 nm 99 nm  94 nm PDI: 0.06 PDI: 0.09 PDI: 0.07 PDI: 0.07 NPA-015-1 DLin-106 nm 100 nm 100 nm  99 nm MC3- PDI: 0.07 PDI: 0.06 PDI: 0.05 PDI: 0.05DMA

TABLE 97 Encapsulation Efficiency Time Formulation # Lipid 0 hours 24hours 48 hours 30 days NPA-003-4 DLin-KC2- 100% 98% 100% 100% DMANPA-006-2 Teta-5-Lap 99% 100% 100% 100% NPA-012-1 DLin-DMA 100% 100%100% 100% NPA-013-1 DLin-K- 83% 85% 96% 100% DMA NPA-014-1 C12-200 88%93% 90% 96% NPA-015-1 DLin-MC3- 100% 99% 100% 100% DMA

Example 67 Intravitreal Delivery

mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 7; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) and luciferasemodified mRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) formulated in salinewas delivered intravitreally in rats as described in Table 98. Thesample was compared against a control of saline only deliveredintravitreally.

TABLE 98 Dosing Chart Dose Level (μg Dose Treatment modified volumeRight Eye Left Eye Group No. RNA/eye) (μL/eye) (OD) (OS) Control 0 5Delivery Delivery buffer only buffer only Modified RNA in 10 5 mCherryLuciferase delivery buffer

The formulation will be administered to the left or right eye of eachanimal on day 1 while the animal is anesthetized. On the day prior toadministration gentamicin ophthalmic ointment or solution was applied toboth eyes twice. The gentamicin ophthalmic ointment or solution was alsoapplied immediately following the injection and on the day following theinjection. Prior to dosing, mydriatic drops (1% tropicamide and/or 2.5%phenylephrine) are applied to each eye.

18 hours post dosing the eyes receiving the dose of mCherry and deliverybuffer are enucleated and each eye was separately placed in a tubecontaining 10 mL 4% paraformaldehyde at room temperature for overnighttissue fixation. The following day, eyes will be separately transferredto tubes containing 10 mL of 30% sucrose and stored at 21° C. until theywere processed and sectioned. The slides prepared from differentsections were evaluated under F-microscopy. Positive expression was seenin the slides prepared with the eyes administered mCherry modified mRNAand the control showed no expression.

Example 68 In Vivo Cytokine Expression Study

Mice were injected intramuscularly with 200 ug of G-CSF modified mRNA(mRNA sequence shown in SEQ ID NO: 6; polyA tail of approximately 160nucleotides not shown in sequence) which was unmodified with a 5′ cap,Cap1 (unmodified), fully modified with 5-methylcytosine andpseudouridine and a 5′ cap, Cap1 (Gen1) or fully modified with5-methylcytosine and N1-methylpseudouridine and a 5′ cap, Cap1 (Gen2cap) or no cap (Gen2 uncapped). Controls of R-848, 5% sucrose anduntreated mice were also analyzed. After 8 hours serum was collectedfrom the mice and analyzed for interferon-alpha (IFN-alpha) expression.The results are shown in Table 99.

TABLE 99 IFN-alpha Expression Formulation IFN-alpha (pg/ml) G-CSFunmodified 67.012 G-CSF Gen1 8.867 G-CSF Gen2 cap 0 G-CSF Gen2 uncapped0 R-848 40.971 5% sucrose 1.493 Untreated 0

Example 69 In Vitro Expression of VEGF Modified mRNA

HEK293 cells were transfected with modified mRNA (mmRNA) VEGF-A (mRNAsequence shown in SEQ ID NO: 19; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) which had been complexed withLipofectamine 2000 from Invitrogen (Carlsbad, Calif.) at theconcentration shown in Table 100. The protein expression was detected byELISA and the protein (pg/ml) is shown in Table 100.

TABLE 100 Protein Expression Amount Transfected 156 10 10 ng 2.5 ng 625pg pg 39 pg pg 2 pg 610 fg Protein 10495 10038 2321.23 189.6 0 0 0 0(pg/ml)

Example 70 In Vitro Screening in HeLa Cells of GFP

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 37.5 ng or 75ng of Green Fluorescent protein (GFP) modified RNA (mRNA sequence shownin SEQ ID NO: 18; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) with the chemical modification described inTable 101, were diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minute at room temperature. Then the 20ul combined solution was added to the 100 ul cell culture mediumcontaining the HeLa cells and incubated at room temperature.

After a 18 to 22 hour incubation cells expressing luciferase were lysedwith 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) accordingto manufacturer instructions. Aliquots of the lysates were transferredto white opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The median fluorescence intensity (MFI) was determinedfor each chemistry and is shown in Table 101.

These results demonstrate that GFP fully modified withN1-methylpseudouridine and 5-methylcytosine produces more protein inHeLa cells compared to the other chemistry. Additionally the higher doseof GFP administered to the cells resulted in the highest MFI value.

TABLE 101 Mean Fluorescence Intensity 37.5 ng 75 ng Chemistry MFI MFI Nomodifications 97400 89500 5-methylcytosine/pseudouridine 324000 7150005-methylcytosine/N1-methylpseudouridine 643000 1990000

Example 71 Toxicity Studies

A. Study Design

Sprague-Dawley rats (n=8, 4 male, 4 female) were administered byinjection modified luciferase mRNA (mRNA sequence shown in SEQ ID NO:16; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) asoutlined in the dosing chart in Table 102. A control group wereadministered the formulation buffer (F. Buffer). After 7 days the ratswere sacrificed.

TABLE 102 Dosing Chart Dose mRNA Dose Dose Volume ConcentrationFormulation (ug) (mL) (mg/mL) Luciferase 100 0.1 0 Luciferase 300 0.11.0 Luciferase 1000 0.1 3.0 Luciferase 3 × 1000 0.3 (each dose 10 was0.1) F. Buffer 0 10

B. Weight Gain and Food Consumption

The rats were weighed before the administration of mRNA and 7 days afteradministration. Table 103 shows the mean weight gain and weight gainpercent per group tested separated by gender. All animals continued togain weight and behave normally. Each group analyzed consumed about thesame amount of food over the course of the study.

TABLE 103 Weight Gain Group Mean weight Gain (g) Weight Gain (%) 100 ug16.875 6.5 300 ug 22.125 8.3 1000 ug 19 6.95 3 × 1000 ug 20.375 7.7 F.Buffer 18.75 6.8

C. Electrolytes

After 7 days the rats were sacrificed and samples were taken todetermine electrolytes. The calcium, bicarbonate, potassium, phosphorus,chloride and sodium levels in each group were analyzed. The results areshown in Table 104. There was no change in electrolytes seen in the ratsafter 7 days.

TABLE 104 Electrolytes Calcium Bicarbonate Potassium Phosphorus ChlorideSodium Group (mg/dL) (mEg/L) (mEg/L) (mg/dL) (mEg/L) (mEg/L) 100 ug 9.819.9 4.7 8.3 101.0 139.6 300 ug 9.8 23.3 4.4 8.2 100.5 139.6 1000 ug10.6 22.5 5.2 9.1 101.0 138.8 3 × 1000 ug 10.2 22.6 4.6 8.11 100.4 138.8F. Buffer 9.6 20.1 5.4 9.2 99.5 139.9

D. Hematology

After 7 days the rats were sacrificed and samples were taken todetermine hematology levels. The red blood cell (RBC), hematocrit (HGT),mean corpuscular volume (MCV), hemoglobin (HGB), mean corpuscularhemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)was determined for each group. The results are shown in Table 105. Therewas no change in blood count or blood clotting factors 7 days afteradministration.

TABLE 105 Hematology RBC HCT MCV HGB MCH MCHC Group (Million/uL) (%)(fL) (g/dL) (pg) (g/dL) 100 ug 7.5 44.1 58.7 14.7 19.5 3.4 300 ug 7.343.5 59.6 14.5 19.8 33.3 1000 ug 7.2 42.5 58.8 14.2 19.55 33.3 3 × 1000ug 7.2 43.5 60.6 14.4 20.0 33.1 F. Buffer 8.0 46.6 58.0 15.5 19.3 33.4

E. White Blood Cells

After 7 days the rats were sacrificed and samples were taken todetermine white blood cell count. Neutrophils (percent segmentedneutrophils), monocytes, basophils, lymphocytes, eosinophil and whiteblood cell (WBC) was determined for each group. The results are shown inTable 106. In Table 106, “NT” means not tested. 7 days afteradministration there was no increase in white blood cells which suggeststhere was no inflammation.

TABLE 106 White Blood Cell Neutrophil WBC (NEU- Monocytes BasophilsLymphocytes Eosinophils (Thous./ Group SEG %) (MON %) (BASO %) (LYM %)(EOS %) uL) 100 ug 10.6 2.0 0.4 85.9 1.3 14 300 ug 12.0 2.8 0.4 83.6 1.010.2 1000 ug 12.8 2.3 NT 83.0 1.5 10.7 3 × 1000 ug 11.6 2.0 0.1 85.5 0.910.9 F. Buffer 16.6 2.3 0.9 79.6 0.9 13.0

F. Serum Chemistry

After 7 days the rats were sacrificed and samples were taken todetermine serum chemistry. The alkaline phosphatase (ALP), aspartatetransaminase (AST), alanine transaminase (ALT) and creatinephosphokinase (CPK) was determined for each group. The results are shownin Table 107.

TABLE 107 Serum Chemistry Group ALP (IU/L) AST (IU/L) ALT (IU/L) CPK(IU/L) 100 ug 144.4 198.3 60.8 488.1 300 ug 169.5 200.3 49.3 968.3 1000ug 150.5 189.8 51.5 744 3 × 1000 ug 152.0 14.3 45.9 481.1 F. Buffer 183170.4 62.8 589.8

G. Liver Proteins

After 7 days the rats were sacrificed and samples were taken todetermine liver protein levels. The level of albumin, globulin and totalprotein was determined for each group. The results are shown in Table108. There was no change seen in liver enzyme or liver proteinproduction 7 days after administration with the modified mRNA.

TABLE 108 Hematology Group Albumin (g/dL) Globulin (g/dL) Total Protein(g/dL) 100 ug 3.3 2.5 5.8 300 ug 3.2 2.4 5.6 1000 ug 3.2 2.7 5.9 3 ×1000 ug 3.4 2.6 6.0 F. Buffer 3.6 2.6 6.2

H. Conclusions

From the analysis of the rats 7 days after administration with themodified mRNA, administration of high doses of mRNA do not result inadverse effects. Doses as high as 30 times the effective dose appear tobe safe from this analysis. Histopathology showed only minimalinflammation at the site of injection and the site of injection showedonly changes consistent with injection and nothing to suggest doserelated issues. Additionally there was no chance in muscle enzymes tosuggest there was muscle damage.

Example 72 Storage Conditions for Modified RNA

A. Organics

To evaluate the ability of mRNA to withstand an organic environment,luciferase mRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was stored at roomtemperature in solutions of ethanol, methanol or dichloromethane at aconcentration of 1 mg/ml. Samples were collected at 1 hour, 6 hours and1 day. The sample was diluted with water to 200 ng/ul and incubatedovernight at room temperature in a fume hood to evaporate off theorganic solvent. Control samples were completed in parallel with mRNA inwater (Water control, organic). The mRNA was stable at room temperaturefor 1 day in each of the three solutions as determined by runningsamples on a bioanalyzer.

B. Aqueous Solvent

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with pseudouridine and 5-methylcytosine) was added to 3different buffers and water to evaluate the effect of aqueous solventson mRNA stability. mRNA was added to citrate buffer (pH3, 100 mM citricacid), phosphate buffered saline (PBS) buffer (pH 7.4, 6.7 mM phosphateand 154 mM sodium chloride), TE buffer (pH 8, 10 mM Tris-hydrochloricacid and 1 mM ethylenediaminetetraacetic acid) or water (pH 5.5, waterfor injection (WFI)) at 1 mg/ml. Samples were collected at 1 hour, 6hours and 1 day and diluted with water to a concentration of 200 ng/ul.Control samples were completed in parallel with mRNA in water (Watercontrol, aqueous). The incubation of mRNA in the PBS buffer, TE bufferand water did not affect the mRNA integrity after 1 day. Samplesincubated in citrate were not detectable by bioanalyzer.

In additional studies to evaluate the citrate buffer, citrate buffer ata pH of 2, 3 and 4 each having 10 mM citrate and 1 mg/ml of luciferasemRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified withpseudouridine and 5-methylcytosine) were evaluated. At a pH of 2precipitation was visually detected and mRNA was not detected bybioanalyzer below a pH of 4. When compared against phosphate buffer,mRNA was not detected in samples with low pH and precipitation wasvisible in phosphate buffer samples having a pH of 2.

C. pH

In order to study the effects of pH on the stability of mRNA, luciferasemRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified withpseudouridine and 5-methylcytosine) was stored at room temperature inaqueous buffers having a pH of 5.8, 6.5 or 7.2. Samples were collectedat 1 hour, 1 day and 1 week after the mRNA was added to the pH sample.After collection the samples were incubated at 1 mg/ml concentration andthen diluted to 200 ng/ul with water before freezing and characterizingby bioanalyzer. The mRNA was stable after 1 week of storage at roomtemperature in the pH range of 5.8-7.2 evaluated.

D. Freeze/Thaw and Lyophilization

To evaluate the effect of freeze/thaw cycles on mRNA stability,luciferase mRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) in formulation bufferwas subjected to numerous freeze/thaw cycles. mRNA was found to bestable for at least 18 cycles.

In addition, luciferase mRNA (mRNA SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was subjected to 3rounds of lyophilization to test the stability of the mRNA. mRNA wasadded to water and samples were collected after each of the 3 rounds oflyophilization. The dried mRNA was diluted with water to reach aconcentration of 1 mg/ml. The samples were stored frozen untilbioanalyzer characterization at 200 ng/ul. Control samples werecompleted in parallel with mRNA and water formulations and followed thesame freezing and thawing cycles. The mRNA was found to be stable after3 cycles of lyophilization when analyzed by bioanalyzer characterizationat 200 ng/ul.

E. Centrifugation

To evaluate the effects of centrifugation on mRNA integrity, luciferasemRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1; 5-methylcytosineand pseudouridine) in water at 1 mg/ml was exposed to 10 cycles of 10 kRPM (13.3 k×g) for 10 minutes at 4° C. mRNA and water samples werestored at 4° C. as a control during centrifugation. After 10 cycles ofcentrifugation the mRNA was still stable when analyzed by bioanalyzercharacterization at 200 ng/ul.

F. In Vitro Transfection after Storage

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 250 ngof luciferase mRNA from the formulations of the lyophilized,centrifuged, organic and aqueous solvent samples were diluted in a 10 ulfinal volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as atransfection reagent and 0.2 ul was diluted in a 10 ul final volume ofOPTI-MEM. After 5 min of incubation at room temperature, both solutionswere combined and incubated an additional 15 min at room temperature.Then 20 ul of the combined solution was added to 100 ul of cell culturemedium containing the HeLa cells. The plates were then incubated asdescribed before.

After 18 h to 22 h incubation, cells expressing luciferase were lysedwith 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.).

Controls of mock transfection (transfection reagent alone), luciferasemRNA control in water, and untreated were also evaluated. Cells wereharvested and the bioluminescence average (in relative light units, RLU)for each signal is shown in Table 109. Transfection of these samplesconfirmed that lyophilization, centrifugation, organic solvents andaqueous solvents except citrate buffer did not impact the activity ofluciferase mRNA. Citrate buffer showed a reduced activity aftertransfection.

TABLE 109 Bioluminescence Sample Bioluminescence (RLU) 1 lyophilization2832350 1 lyophilization control 3453250 2 lyophilizations 2480000 2lyophilizations control 3716130 3 lyophilizations 1893960 3lyophilizations control 3009020 Centrifugation, 10 cycles 3697590Centrifugation control 5472920 Ethanol, 1 day 4214780 Methanol, 1 day2834520 Dichloromethane, 1 day 3017890 Water control, organic, 1 day2641450 Citrate buffer, 1 hour 280160 PBS buffer, 1 hour 2762050 TEbuffer, 1 hour 3141250 Water control, aqueous, 1 hour 3394000 Citratebuffer, 1 day 269790 PBS buffer, 1 day 4084330 TE buffer, 1 day 5344400Water control, aqueous, 1 day 3579270 Untreated 5580 Mock Transfection7560 Luciferase mRNA control 4950090

Example 73 Homogenization

Different luciferase mRNA solutions (as described in Table 110 where “X”refers to the solution containing that component) (mRNA sequence shownin SEQ ID NO: 16; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) were evaluated to test the percent yield of the differentsolutions, the integrity of the mRNA by bioanalyzer, and the proteinexpression of the mRNA by in vitro transfection. The mRNA solutions wereprepared in water, 1×TE buffer at 4 mg/ml as indicated in Table 110, andadded to either dichloromethane (DCM) or DCM containing 200 mg/ml ofpoly(lactic-co-glycolic acid) (PLGA) (Lactel, Cat# B6010-2, inherentviscosity 0.55-0.75, 50:50 LA:GA) to achieve a final mRNA concentrationof 0.8 mg/ml. The solutions requiring homogenization were homogenizedfor 30 seconds at speed 5 (approximately 19,000 rpm) (IKA Ultra-TurraxHomogenizer, T18). The mRNA samples in water, dichloromethane andpoly(lactic-co-glycolic acid) (PLGA) were not recoverable (NR). Allsamples, except the NR samples, maintained integrity of the mRNA asdetermined by bioanalyzer (Bio-rad Experion).

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 250 ngof luciferase mRNA from the recoverable samples was diluted in a 10 ulfinal volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as atransfection reagent and 0.2 ul was diluted in a 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 20 ul of the combined solution was added to 100 ul ofcell culture medium containing the HeLa cells. The plates were thenincubated as described before. Controls luciferase mRNA (luciferase mRNAformulated in saline) (Control) and untreated cells (Untreat.) were alsoevaluated. Cells were harvested and the bioluminescence average (inphotons/second) (biolum. (p/s)) for each signal is also shown in Table110. The recoverable samples all showed activity of luciferase mRNA whenanalyzed.

After 18 to 22 hour incubation, cells expressing luciferase were lysedwith 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.).

Cells were harvested and the bioluminescence average (in relative lightunits, RLU) (biolum. (RLU)) for each signal is also shown in Table 110.The recoverable samples all showed activity of luciferase mRNA whenanalyzed.

TABLE 110 Solutions Solution 1× TE DCM/ Homo- Yield Biolum. No. WaterBuffer DCM PLGA genizer (%) (RLU) 1 X 96 5423780 2 X X 95 4911950 3 X X92 2367230 4 X X 90 4349410 5 X X X 66 4145340 6 X X X 71 3834440 7 X XX NR n/a 8 X X X 24 3182080 9 X X NR n/a 10  X X 79 3276800 11  X X 795563550 12  X X 79 4919100 Control 2158060 Untreat.   3530

Example 74 TE Buffer and Water Evaluation

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 16; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was reconstituted inwater or TE buffer as outlined in Table 111 and then formulated in PLGAmicrospheres. PLGA microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23 k)dichloromethane and water. Briefly, 0.2 to 0.6 ml of mRNA in water or TEbuffer at a concentration of 2 to 6 mg/ml (W1) was added to 2 ml of PLGAdissolved in dichloromethane (DCM) (O1) at a concentration of 100 mg/mlof PLGA. The W1/O1 emulsion was homogenized (IKA Ultra-TurraxHomogenizer, T18) for 30 seconds at speed 5 (˜19,000 rpm). The W1/O1emulsion was then added to 250 ml 1% PVA (W2) and homogenized for 1minute at speed 5 (˜19,000 rpm). Formulations were left to stir for 3hours, then passed through a 100 μm nylon mesh strainer (FisherbrandCell Strainer, Cat #22-363-549) to remove larger aggregates, and finallywashed by centrifugation (10 min, 9,250 rpm, 4° C.). The supernatant wasdiscarded and the PLGA pellets were resuspended in 5-10 ml of water,which was repeated 2×. The washed formulations were frozen in liquidnitrogen and then lyophilized for 2-3 days. After lyophilization, ˜10 mgof PLGA MS were weighed out in 2 ml eppendorf tubes and deformulated byadding 1 ml of DCM and letting the samples shake for 2-6 hrs. mRNA wasextracted from the deformulated PLGA micropsheres by adding 0.5 ml ofwater and shaking the sample overnight. Unformulated luciferase mRNA inwater or TE buffer (deformulation controls) was spiked into DCM and wentthrough the deformulation process to be used as controls in thetransfection assay.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 100 ngof the deformulated luciferase mRNA samples was diluted in a 1 0 ulfinal volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as atransfection reagent and 0.2 ul was diluted in a 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 20 ul of the combined solution was added to 100 ul ofcell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After 18 to 22 hour incubation, cells expressing luciferase were lysedwith 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.). To determine the activity of theluciferase mRNA from each formulation, the relative light units (RLU)for each formulation was divided by the RLU of the appropriate mRNAdeformulation control (mRNA in water or TE buffer). Table 111 shows theactivity of the luciferase mRNA. The activity of the luciferase mRNA inthe PLGA microsphere formulations (Form.) was substantially improved byformulating in TE buffer versus water.

TABLE 111 Formulations W1 Theoretical Actual mRNA Solvent Total mRNAmRNA Activity (% of conc. volume mRNA Loading Loading W1 deformulationForm. (mg/ml) (ul) (ug) (wt %) (wt %) Solvent control) PLGA A 4 400 16000.80 0.14 Water 12.5% PLGA B 4 200 800 0.40 0.13 Water 1.3% PLGA C 4 6002400 1.20 0.13 Water 12.1% PLGA D 2 400 800 0.40 0.07 Water 1.3% PLGA E6 400 2400 1.20 0.18 TE 38.9% Buffer PLGA F 4 400 1600 0.80 0.16 TE39.7% Buffer PLGA G 4 400 1600 0.80 0.10 TE 26.6% Buffer

Example 75 Chemical Modifications on mRNA

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(Life Technologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown SEQ ID NO: 16; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1)with the chemical modification described in Table 112, were diluted in10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 112. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.).The background signal of theplates without reagent was about 200 relative light units per well.

TABLE 112 Chemical Modifications Sample RLU Untreated 336 UnmodifiedLuciferase 33980 5-methylcytosine and pseudouridine 16012345-methylcytosine and N1-methylpseudouridine 421189 25% cytosinesreplaced with 5-methylcytosine and 222114 25% of uridines replaced with2-thiouridine N1-methylpseudouridine 3068261 Pseudouridine 140234N4-Acetylcytidine 1073251 5-methoxyuridine 219657 5-Bromouridine 6787N4-Acetylcytidine and N1-methylpseudouridine 976219 5-methylcytosine and5-methoxyuridine 66621 5-methylcytosine and 2′fluorouridine 11333

Example 76 Intramuscular and Subcutaneous Administration of ModifiedmRNA

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 16; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 5-methylcytosine and pseudouridine (5mC/pU),fully modified with 5-methylcytosine and N1-methylpseudouridine(5mC/N1mpU), fully modified with pseudouridine (pU), fully modified withN1-methylpseudouridine (N1mpU) or modified where 25% of the cytosinesreplaced with 5-methylcytosine and 25% of the uridines replaced with2-thiouridine (5mC/s2U) formulated in PBS (pH 7.4) was administered toBalb-C mice intramuscularly or subcutaneously at a dose of 2.5 mg/kg.The mice were imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours,96 hours, 120 hours and 144 hours for intramuscular delivery and 2hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours forsubcutaneous delivery. Twenty minutes prior to imaging, mice wereinjected intraperitoneally with a D-luciferin solution at 150 mg/kg.Animals were then anesthetized and images were acquired with an IVISLumina II imaging system (Perkin Elmer). Bioluminescence was measured astotal flux (photons/second) of the entire mouse. The average total flux(photons/second) for intramuscular administration is shown in Table 113and the average total flux (photons/second) for subcutaneousadministration is shown in Table 114. The background signal was 3.79E+05(p/s). The peak expression for intramuscular administration was seenbetween 24 and 48 hours for all chemistry and expression was stilldetected at 144 hours. For subcutaneous delivery the peak expression wasseen at 2-8 hours and expression was detected at 72 hours.

TABLE 113 Intramuscular Administration 5mC/ 5mC/pU N1mpU 5mC/s2U pUN1mpU Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hours1.98E+07 4.65E+06 4.68E+06 2.33E+06 3.66E+07  8 hours 1.42E+07 3.64E+063.78E+06 8.07E+06 7.21E+07 24 hours 2.92E+07 1.22E+07 3.35E+07 1.01E+071.75E+08 48 hours 2.64E+07 1.01E+07 5.06E+07 7.46E+06 3.42E+08 72 hours2.18E+07 8.59E+06 3.42E+07 4.08E+06 5.83E+07 96 hours 2.75E+07 2.70E+062.38E+07 4.35E+06 7.15E+07 120 hours  2.19E+07 1.60E+06 1.54E+071.25E+06 3.87E+07 144 hours  9.17E+06 2.19E+06 1.14E+07 1.86E+065.04E+07

TABLE 114 Subcutaneous Administration 5mC/ 5mC/pU N1mpU 5mC/s2U pU N1mpUFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hours 5.26E+064.54E+06 9.34E+06 2.43E+06 2.80E+07  8 hours 2.32E+06 8.75E+05 8.15E+062.12E+06 3.09E+07 24 hours 2.67E+06 5.49E+06 3.80E+06 2.24E+06 1.48E+0748 hours 1.22E+06 1.77E+06 3.07E+06 1.58E+06 1.24E+07 72 hours 1.12E+068.00E+05 8.53E+05 4.80E+05 2.29E+06 96 hours 5.16E+05 5.33E+05 4.30E+054.30E+05 6.62E+05 120 hours  3.80E+05 4.09E+05 3.21E+05 6.82E+055.05E+05

Example 77 Osmotic Pump Study

Prior to implantation, an osmotic pump (ALZET® Osmotic Pump 2001D,DURECT Corp. Cupertino, Calif.) is loaded with the 0.2 ml of 1×PBS (pH7.4) (PBS loaded pump) or 0.2 ml of luciferase modified mRNA (mRNAsequence shown in SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and N1-methylpseudouridine) at 1 mg/ml in 1×PBS (pH7.4) (Luciferase loaded pump) and incubated overnight in 1×PBS (pH 7.4)at 37° C.

Balb-C mice (n=3) are implanted subcutaneously with either the PBSloaded pump or the luciferase loaded pump and imaged at 2 hours, 8 hoursand 24 hours. As a control a PBS loaded pump is implanted subcutaneouslyand the mice are injected subcutaneously with luciferase modified mRNAin 1×PBS (PBS loaded pump; SC Luciferase) or an osmotic pump is notimplanted and the mice are injected subcutaneously with luciferasemodified mRNA in 1×PBS (SC Luciferase). The luciferase formulations areoutlined in Table 115

TABLE 115 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) PBS loaded pump; SC PBS 1.00 50 50 2.5Luciferase Luciferase loaded pump PBS 1.00 — 200  10.0  PBS loaded pumpPBS — — — — SC Luciferase PBS 1.00 50 50 2.5

Example 78 External Osmotic Pump Study

An external osmotic pump (ALZET® Osmotic Pump 2001D, DURECT Corp.Cupertino, Calif.) is loaded with the 0.2 ml of 1×PBS (pH 7.4) (PBSloaded pump) or 0.2 ml of luciferase modified mRNA (mRNA sequence shownin SEQ ID NO: 16; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andN1-methylpseudouridine) at 1 mg/ml in 1×PBS (pH 7.4) (luciferase loadedpump) and incubated overnight in 1×PBS (pH 7.4) at 37° C.

Using a catheter connected to the external PBS loaded pump or theluciferase loaded pump Balb-C mice (n=3) are administered theformulation. The mice are imaged at 2 hours, 8 hours and 24 hours. As acontrol an external PBS loaded pump is used and the mice are injectedsubcutaneously with luciferase modified mRNA in 1×PBS (PBS loaded pump;SC Luciferase) or the external pump is not used and the mice are onlyinjected subcutaneously with luciferase modified mRNA in 1×PBS (SCLuciferase). Twenty minutes prior to imaging, mice are injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals arethen anesthetized and images are acquired with an IVIS Lumina II imagingsystem (Perkin Elmer). Bioluminescence is measured as total flux(photons/second) of the entire mouse. The luciferase formulations areoutlined in Table 116 and the average total flux (photons/second).

TABLE 116 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) PBS loaded pump; SC PBS 1.00 50 50 2.5Luciferase Luciferase loaded pump PBS 1.00 — 200  10.0  PBS loaded pumpPBS — — — — SC Luciferase PBS 1.00 50 50 2.5

Example 79 Fibrin Sealant Study

Fibrin sealant, such as Tisseel (Baxter Healthcare Corp., Deerfield,Ill.), is composed of fibrinogen and thrombin in a dual-barreledsyringe. Upon mixing, fibrinogen is converted to fibrin to form a fibrinclot in about 10 to 30 seconds. This clot can mimic the natural clottingmechanism of the body. Additionally a fibrin hydrogel is a threedimensional structure that can potentially be used in sustained releasedelivery. Currently, fibrin sealant is approved for application inhemostasis and sealing to replace conventional surgical techniques suchas suture, ligature and cautery.

The thrombin and fibrinogen components were loaded separately into adual barreled syringe. Balb-C mice (n=3) were injected subcutaneouslywith 50 ul of fibrinogen, 50 ul of thrombin and they were also injectedat the same site with modified luciferase mRNA (mRNA sequence shown inSEQ ID NO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andN1-methylpseudouridine) (Tisseel+Luciferase), 50 ul of fibrinogen and 50ul thrombin (Tisseel) or modified luciferase mRNA (Luciferase). Theinjection of fibrinogen and thrombin was done simultaneously using thedual-barreled syringe. The subcutaneous injection of luciferase was done15 minutes after the fibrinogen/thrombin injection to allow the fibrinhydrogel to polymerize (Tisseel+Luciferase group). A control group ofuntreated mice were also evaluated. The mice were imaged at 5 hours and24 hours. Twenty minutes prior to imaging, mice were injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals werethen anesthetized and images were acquired with an IVIS Lumina IIimaging system (Perkin Elmer). Bioluminescence was measured as totalflux (photons/second) of the entire mouse. The luciferase formulationsare outlined in Table 117 and the average total flux (photons/second) isshown in Table 118. The fibrin sealant was found to not interfere withimaging and the injection of luciferase and Tisseel showed expression ofluciferase.

TABLE 117 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) Tisseel + Luciferase PBS 1.00 50 50 2.5Tisseel — — — — — Luciferase PBS 1.00 50 50 2.5 Untreated — — — — —

TABLE 118 Total Flux 5 Hours 24 Hours Group Flux (p/s) Flux (p/s)Tisseel + Luciferase 4.59E+05 3.39E+05 Tisseel 1.99E+06 1.06E+06Luciferase 9.94E+05 7.44E+05 Untreated 3.90E+05 3.79E+05

Example 80 Fibrin Containing mRNA Sealant Study

A. Modified mRNA and Calcium Chloride

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methylpseudouridine isadded to calcium chloride. The calcium chloride is then used toreconstitute thrombin. Fibrinogen is reconstituted with fibrinolysisinhibitor solution per the manufacturer's instructions. Thereconstituted thrombin containing modified mRNA and fibrinogen is loadedinto a dual barreled syringe. Mice are injected subcutaneously with 50ul of fibrinogen and 50 ul of thrombin containing modified mRNA or theywere injected with 50 ul of PBS containing an equivalent dose ofmodified luciferase mRNA. A control group of untreated mice is alsoevaluated. The mice are imaged at predetermined intervals to determinethe average total flux (photons/second).

B. Lipid Nanoparticle Formulated Modified mRNA and Calcium Chloride

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methylpseudouridine isformulated in a lipid nanoparticle is added to calcium chloride. Thecalcium chloride is then used to reconstitute thrombin. Fibrinogen isreconstituted with fibrinolysis inhibitor solution per themanufacturer's instructions. The reconstituted thrombin containingmodified mRNA and fibrinogen is loaded into a dual barreled syringe.Mice are injected subcutaneously with 50 ul of fibrinogen and 50 ul ofthrombin containing modified mRNA or they were injected with 50 ul ofPBS containing an equivalent dose of modified luciferase mRNA. A controlgroup of untreated mice is also evaluated. The mice are imaged atpredetermined intervals to determine the average total flux(photons/second).

C. Modified mRNA and Fibrinogen

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methylpseudouridine isadded to the fibrinolysis inhibitor solution. The fibrinolysis inhibitorsolution is then used to reconstitute fibrinogen. Thrombin isreconstituted with the calcium chloride solution per the manufacturer'sinstructions. The reconstituted fibrinogen containing modified mRNA andthrombin is loaded into a dual barreled syringe. Mice are injectedsubcutaneously with 50 ul of thrombin and 50 ul of fibrinogen containingmodified mRNA or they were injected with 50 ul of PBS containing anequivalent dose of modified luciferase mRNA. A control group ofuntreated mice is also evaluated. The mice are imaged at predeterminedintervals to determine the average total flux (photons/second).

D. Lipid Nanoparticle Formulated Modified mRNA and Fibrinogen

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methylpseudouridine isformulated in a lipid nanoparticle is added to the fibrinolysisinhibitor solution. The fibrinolysis inhibitor solution is then used toreconstitute fibrinogen. Thrombin is reconstituted with the calciumchloride solution per the manufacturer's instructions. The reconstitutedfibrinogen containing modified mRNA and thrombin is loaded into a dualbarreled syringe. Mice are injected subcutaneously with 50 ul ofthrombin and 50 ul of fibrinogen containing modified mRNA or they wereinjected with 50 ul of PBS containing an equivalent dose of modifiedluciferase mRNA. A control group of untreated mice is also evaluated.The mice are imaged at predetermined intervals to determine the averagetotal flux (photons/second).

E. Modified mRNA and Thrombin

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methylpseudouridine isadded to the reconstituted thrombin after it is reconstituted with thecalcium chloride per the manufacturer's instructions. The fibrinolysisinhibitor solution is then used to reconstitute fibrinogen per themanufacturer's instructions. The reconstituted fibrinogen and thrombincontaining modified mRNA is loaded into a dual barreled syringe. Miceare injected subcutaneously with 50 ul of thrombin containing modifiedmRNA and 50 ul of fibrinogen or they were injected with 50 ul of PBScontaining an equivalent dose of modified luciferase mRNA. A controlgroup of untreated mice is also evaluated. The mice are imaged atpredetermined intervals to determine the average total flux(photons/second).

F. Lipid Nanoparticle Formulated Modified mRNA and Thrombin

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 16; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methylpseudouridine isformulated in a lipid nanoparticle is added to the reconstitutedthrombin after it is reconstituted with the calcium chloride per themanufacturer's instructions. The fibrinolysis inhibitor solution is thenused to reconstitute fibrinogen per the manufacturer's instructions. Thereconstituted fibrinogen and thrombin containing modified mRNA is loadedinto a dual barreled syringe. Mice are injected subcutaneously with 50ul of thrombin containing modified mRNA and 50 ul of fibrinogen or theywere injected with 50 ul of PBS containing an equivalent dose ofmodified luciferase mRNA. A control group of untreated mice is alsoevaluated. The mice are imaged at predetermined intervals to determinethe average total flux (photons/second).

Example 81 Cationic Lipid Formulation of 5-Methylcytosine andN1-Methylpseudouridine Modified mRNA

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and N1-methylpseudouridine was formulated in thecationic lipids described in Table 119. The formulations wereadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 119 Cationic Lipid Formulations Formulation NPA-126-1 NPA-127-1NPA-128-1 NPA-129-1 111612-B Lipid DLin-MC3- DLin-KC2- C12-200 DLinDMADODMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio (wt/wt) MeanSize 122 nm 114 nm 153 nm 137 nm 223.2 nm PDI: 0.13 PDI: 0.10 PDI: 0.17PDI: 0.09 PDI: 0.142 Zeta at pH 7.4 −1.4 mV −0.5 mV −1.4 mV 2.0 mV −3.09mV Encaps. 95% 77% 69% 80% 64% (RiboGr)

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 4.17E+05 p/s. The results of the imaging areshown in Table 120. In Table 120, “NT” means not tested.

TABLE 120 Flux DLin-MC3- DLin-KC2- DMA DMA C12-200 DLinDMA DODMA RouteTime Point Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2hrs 1.92E+08 2.91E+08 1.08E+08 2.53E+07 8.40E+06 I.V. 8 hrs 1.47E+082.13E+08 3.72E+07 3.82E+07 5.62E+06 I.V. 24 hrs  1.32E+07 2.41E+075.35E+06 4.20E+06 8.97E+05 I.M. 2 hrs 8.29E+06 2.37E+07 1.80E+071.51E+06 NT I.M. 8 hrs 5.83E+07 2.12E+08 2.60E+07 1.99E+07 NT I.M. 24hrs  4.30E+06 2.64E+07 3.01E+06 9.46E+05 NT S.C. 2 hrs 1.90E+07 5.16E+078.91E+07 4.66E+06 9.61E+06 S.C. 8 hrs 7.74E+07 2.00E+08 4.58E+079.67E+07 1.90E+07 S.C. 24 hrs  7.49E+07 2.47E+07 6.96E+06 6.50E+061.28E+06

Example 82 Lipid Nanoparticle Intravenous Study

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) was formulated in a lipidnanoparticle containing 50% DLin-MC3-DMA OR DLin-KC2-DMA as described inTable 121, 38.5% cholesterol, 10% DSPC and 1.5% PEG. The formulation wasadministered intravenously (I.V.) to Balb-C mice at a dose of 0.5 mg/kg,0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg. Twenty minutes prior toimaging, mice were injected intraperitoneally with a D-luciferinsolution at 150 mg/kg. Animals were then anesthetized and images wereacquired with an IVIS Lumina II imaging system (Perkin Elmer).Bioluminescence was measured as total flux (photons/second) of theentire mouse.

TABLE 121 Formulations Formulation NPA-098-1 NPA-100-1 LipidDLin-KC2-DMA DLin-MC3-DMA Lipid/mRNA ratio (wt/wt) 20:1 20:1 Mean Size135 nm 152 nm PDI: 0.08 PDI: 0.08 Zeta at pH 7.4 −0.6 mV −1.2 mV Encaps.(RiboGr) 91% 94%

For DLin-KC2-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 72hours, 96 hours and 168 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 3.66E+05 p/s.The results of the imaging are shown in Table 122. Organs were imaged at8 hours and the average total flux (photons/second) was measured for theliver, spleen, lung and kidney. A control for each organ was alsoanalyzed. The results are shown in Table 123. The peak signal for alldose levels was at 8 hours after administration. Also, distribution tothe various organs (liver, spleen, lung, and kidney) may be able to becontrolled by increasing or decreasing the LNP dose.

TABLE 122 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg 0.0005 mg/kg Time PointFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hrs 3.54E+08 1.75E+072.30E+06 4.09E+05  8 hrs 1.67E+09 1.71E+08 9.81E+06 7.84E+05 24 hrs2.05E+08 2.67E+07 2.49E+06 5.51E+05 72 hrs 8.17E+07 1.43E+07 1.01E+063.75E+05 96 hrs 4.10E+07 9.15E+06 9.58E+05 4.29E+05 168 hrs  3.42E+079.15E+06 1.47E+06 5.29E+05

TABLE 123 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s)   0.5 mg/kg 1.42E+08 4.86E+07 1.90E+05 3.20E+05  0.05mg/kg 7.45E+06 4.62E+05 6.86E+04 9.11E+04  0.005 mg/kg 3.32E+05 2.97E+041.42E+04 1.15E+04 0.0005 mg/kg 2.34E+04 1.08E+04 1.87E+04 9.78E+03Untreated 1.88E+04 1.02E+04 1.41E+04 9.20E+03

For DLin-MC3-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 48hours, 72 hours and 144 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 4.51E+05 p/s.The results of the imaging are shown in Table 124. Organs were imaged at8 hours and the average total flux (photons/second) was measured for theliver, spleen, lung and kidney. A control for each organ was alsoanalyzed. The results are shown in Table 125. The peak signal for alldose levels was at 8 hours after administration. Also, distribution tothe various organs (liver, spleen, lung, and kidney) may be able to becontrolled by increasing or decreasing the LNP dose.

TABLE 124 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg 0.0005 mg/kg Time PointFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hrs 1.23E+08 7.76E+067.66E+05 4.88E+05  8 hrs 1.05E+09 6.79E+07 2.75E+06 5.61E+05 24 hrs4.44E+07 1.00E+07 1.06E+06 5.71E+05 48 hrs 2.12E+07 4.27E+06 7.42E+054.84E+05 72 hrs 1.34E+07 5.84E+06 6.90E+05 4.38E+05 144 hrs  4.26E+062.25E+06 4.58E+05 3.99E+05

TABLE 125 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s)   0.5 mg/kg 1.19E+08 9.66E+07 1.19E+06 1.85E+05  0.05mg/kg 1.10E+07 1.79E+06 7.23E+04 5.82E+04  0.005 mg/kg 3.58E+05 6.04E+041.33E+04 1.33E+04 0.0005 mg/kg 2.25E+04 1.88E+04 2.05E+04 1.65E+04Untreated 1.91E+04 1.66E+04 2.63E+04 2.14E+04

Example 83 Lipid Nanoparticle Subcutaneous Study

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) was formulated in a lipidnanoparticle containing 50% DLin-KC2-DMA as described in Table 126, 385%cholesterol, 10% DSPC and 1.5% PEG. The formulation was administeredsubcutaneously (S.C.) to Balb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kgor 0.005 mg/kg.

TABLE 126 DLin-KC2-DMA Formulation Formulation NPA-098-1 LipidDLin-KC2-DMA Lipid/mRNA ratio (wt/wt) 20:1 Mean Size 135 nm PDI: 0.08Zeta at pH 7.4 −0.6 mV Encaps. (RiboGr) 91%

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours, 24 hours, 48hours, 72 hours and 144 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The lower limit of detection was about 3E+05p/s. The results of the imaging are shown in Table 127. Organs wereimaged at 8 hours and the average total flux (photons/second) wasmeasured for the liver, spleen, lung and kidney. A control for eachorgan was also analyzed. The results are shown in Table 128. The peaksignal for all dose levels was at 8 hours after administration. Also,distribution to the various organs (liver, spleen, lung, and kidney) maybe able to be controlled by increasing or decreasing the LNP dose. Athigh doses, the LNP formulations migrates outside of the subcutaneousinjection site, as high levels of luciferase expression are detected inthe liver, spleen, lung, and kidney.

TABLE 127 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg Time Point Flux (p/s)Flux (p/s) Flux (p/s)  2 hrs 3.18E+07 7.46E+06 8.94E+05  8 hrs 5.15E+082.18E+08 1.34E+07 24 hrs 1.56E+08 5.30E+07 7.16E+06 48 hrs 5.22E+078.75E+06 9.06E+05 72 hrs 8.87E+06 1.50E+06 2.98E+05 144 hrs  4.55E+053.51E+05 2.87E+05

TABLE 128 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s)  0.5 mg/kg 1.01E+07 7.43E+05 9.75E+04 1.75E+05  0.05mg/kg 1.61E+05 3.94E+04 4.04E+04 3.29E+04 0.005 mg/kg 2.84E+04 2.94E+042.42E+04 9.79E+04 Untreated 1.88E+04 1.02E+04 1.41E+04 9.20E+03

Example 84 Catioinic Lipid Nanoparticle Subcutaneous Study

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) is formulated in a lipidnanoparticle containing 50% DLin-MC3-DMA, 38.5% cholesterol, 10% DSPCand 1.5% PEG. The formulation is administered subcutaneously (S.C.) toBalb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg.

The mice are imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hoursand 144 hours after dosing and the average total flux (photons/second)was measured for each route of administration and cationic lipidformulation. Organs are imaged at 8 hours and the average total flux(photons/second) is measured for the liver, spleen, lung and kidney. Acontrol for each organ is also analyzed.

Example 85 Lipoplex Study

Lipoplexed luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately140 nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and N1-methylpseudouridine (5mC/N1mpU) or modifiedwhere 25% of the cytosines replaced with 5-methylcytosine and 25% of theuridines replaced with 2-thiouridine (5mC/s2U). The formulation wasadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.10 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 8 hours, 24 hours and 48 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and chemical modification. Thebackground signal was about 3.91E+05 p/s. The results of the imaging areshown in Table 129. Organs were imaged at 6 hours and the average totalflux (photons/second) was measured for the liver, spleen, lung andkidney. A control for each organ was also analyzed. The results areshown in Table 130.

TABLE 129 Flux Time 5mC/pU 5mC/N1mpU 5mC/s2U Route Point Flux (p/s) Flux(p/s) Flux (p/s) I.V.  8 hrs 5.76E+06 1.78E+06 1.88E+06 I.V. 24 hrs1.02E+06 7.13E+05 5.28E+05 I.V. 48 hrs 4.53E+05 3.76E+05 4.14E+05 I.M. 8 hrs 1.90E+06 2.53E+06 1.29E+06 I.M. 24 hrs 9.33E+05 7.84E+05 6.48E+05I.M. 48 hrs 8.51E+05 6.59E+05 5.49E+05 S.C.  8 hrs 2.85E+06 6.48E+061.14E+06 S.C. 24 hrs 6.66E+05 7.15E+06 3.93E+05 S.C. 48 hrs 3.24E+053.20E+06 5.45E+05

TABLE 130 Organ Flux Liver Spleen Lung Kidney Inj. Site Route ChemistryFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 5mC/pU5.26E+05 2.04E+07 4.28E+06 1.77E+04 n/a I.V. 5mC/N1mpU 1.48E+05 5.00E+061.93E+06 1.77E+04 n/a I.V. 5mC/s2U 2.14E+04 3.29E+06 5.48E+05 2.16E+04n/a I.M. 5mC/pU 2.46E+04 1.38E+04 1.50E+04 1.44E+04 1.15E+06 I.M.5mC/N1mpU 1.72E+04 1.76E+04 1.99E+04 1.56E+04 1.20E+06 I.M. 5mC/s2U1.28E+04 1.36E+04 1.33E+04 1.07E+04 7.60E+05 S.C. 5mC/pU 1.55E+041.67E+04 1.45E+04 1.69E+04 4.46E+04 S.C. 5mC/N1mpU 1.20E+04 1.46E+041.38E+04 1.14E+04 8.29E+04 S.C. 5mC/s2U 1.22E+04 1.31E+04 1.45E+041.08E+04 5.62E+04 Untreated 2.59E+04 1.34E+04 1.26E+04 1.22E+04 n/a

Example 86 Cationic Lipid Formulation of Modified mRNA

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) modified where 25% ofthe cytosines replaced with 5-methylcytosine and 25% of the uridinesreplaced with 2-thiouridine (5mC/s2U) was formulated in the cationiclipids described in Table 131. The formulations were administeredintravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) toBalb-C mice at a dose of 0.05 mg/kg.

TABLE 131 Cationic Lipid Formulations Formulation NPA-130-1 NPA-131-1NPA-132-1 NPA-133-1 111612-C Lipid DLin-MC3- DLin-KC2- C12-200 DLinDMADODMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio (wt/wt) MeanSize 120 nm 105 nm 122 nm 105 nm 221.3 nm PDI: 0.10 PDI: 0.11 PDI: 0.13PDI: 0.14 PDI: 0.063 Zeta at pH 7.4 0.2 mV −0.6 mV −0.5 mV −0.3 mV −3.10mV Encaps. 100% 100% 93% 93% 60% (RiboGr)

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 3.31E+05 p/s. The results of the imaging areshown in Table 132. In Table 132, “NT” means not tested. Untreated miceshowed an average flux of 3.14E+05 at 2 hours, 3.33E+05 at 8 hours and3.46E+05 at 24 hours. Peak expression was seen for all three routestested at 8 hours. DLin-KC2-DMA has better expression than DLin-MC3-DMAand DODMA showed expression for all routes evaluated.

TABLE 132 Flux DLin-MC3- DLin-KC2- DMA DMA C12-200 DLinDMA DODMA RouteTime Point Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2hrs 9.88E+06 6.98E+07 9.18E+06 3.98E+06 5.79E+06 I.V. 8 hrs 1.21E+071.23E+08 1.02E+07 5.98E+06 6.14E+06 I.V. 24 hrs  2.02E+06 1.05E+071.25E+06 1.35E+06 5.72E+05 I.M. 2 hrs 6.72E+05 3.66E+06 3.25E+067.34E+05 4.42E+05 I.M. 8 hrs 7.78E+06 2.85E+07 4.29E+06 2.22E+061.38E+05 I.M. 24 hrs  4.22E+05 8.79E+05 5.95E+05 8.48E+05 4.80E+05 S.C.2 hrs 2.37E+06 4.77E+06 4.44E+06 1.07E+06 1.05E+06 S.C. 8 hrs 3.65E+071.17E+08 3.71E+06 9.33E+06 2.57E+06 S.C. 24 hrs  4.47E+06 1.28E+076.39E+05 8.89E+05 4.27E+05

Example 87 Formulation of 5-Methylcytosine and N1-MethylpseudouridineModified mRNA

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and N1-methylpseudouridine was formulated in PBS (pH of7.4). The formulations were administered intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 2.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 5 minutes, 30 minutes, 60minutes and 120 minutes after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 3.78E+05 p/s.The results of the imaging are shown in Table 133. Expression ofluciferase was already seen at 30 minutes with both routes of delivery.Peak expression from subcutaneous administration appears between 30 to60 minutes. Intramuscular expression was still increasing at 120minutes.

TABLE 133 Flux PBS (pH 7.4) Route Time Point Flux (p/s) I.M.  5 min4.38E+05 I.M. 30 min 1.09E+06 I.M. 60 min 1.18E+06 I.M. 120 min 2.86E+06 S.C.  5 min 4.19E+05 S.C. 30 min 6.38E+06 S.C. 60 min 5.61E+06S.C. 120 min  2.66E+06

Example 88 Intramuscular and Subcutaneous Administration of ChemicallyModified mRNA

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 16; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with N4-acetylcytidine, fully modified with5-methoxyuridine, fully modified with N4-acetylcytidine andN1-methylpseudouridine or fully modified 5-methylcytosine and5-methoxyuridine formulated in PBS (pH 7.4) was administered to Balb-Cmice intramuscularly or subcutaneously at a dose of 2.5 mg/kg. Twentyminutes prior to imaging, mice were injected intraperitoneally with aD-luciferin solution at 150 mg/kg. Animals were then anesthetized andimages were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours.The average total flux (photons/second) for intramuscular administrationis shown in Table 134 and the average total flux (photons/second) forsubcutaneous administration is shown in Table 135. The background signalwas 3.84E+05 (p/s). The peak expression for intramuscular administrationwas seen between 24 and 48 hours for all chemistry and expression wasstill detected at 120 hours. For subcutaneous delivery the peakexpression was seen at 2-8 hours and expression was detected at 72hours.

TABLE 134 Intramuscular Administration 2 hours 8 hours 24 hours Flux(p/s) Flux (p/s) Flux (p/s) N4-acetylcytidine 1.32E+07 2.15E+07 4.01E+075-methoxyuridine 4.93E+06 1.80E+07 4.53E+07 N4-acetylcytidine/ 2.02E+071.93E+07 1.63E+08 N1-methylpseudouridine 5-methylcytosine/5- 6.79E+064.55E+07 3.44E+07 methoxyuridine

TABLE 135 Subcutaneous Administration 2 hours 8 hours 24 hours Flux(p/s) Flux (p/s) Flux (p/s) N4-acetylcytidine 3.07E+07 1.23E+07 1.28E+075-methoxyuridine 7.10E+06 9.38E+06 1.32E+07 N4-acetylcytidine/ 7.12E+063.07E+06 1.03E+07 N1-methylpseudouridine 5-methylcytosine/5- 7.15E+061.25E+07 1.11E+07 methoxyuridine

Example 89 In Vivo Study

Luciferase modified mRNA containing at least one chemical modificationis formulated as a lipid nanoparticle (LNP) using the syringe pumpmethod and characterized by particle size, zeta potential, andencapsulation.

As outlined in Table 136, the luciferase LNP formulation is administeredto Balb-C mice intramuscularly (I.M.), intravenously (I.V.) andsubcutaneously (S.C.). As a control luciferase modified RNA formulatedin PBS is administered intravenously to mice.

TABLE 136 Luciferase Formulations Concen- Injection Amount of trationVolume modified Dose Formulation Vehicle Route (mg/ml) (ul) RNA (ug)(mg/kg) Luc-LNP PBS S.C. 0.2000 50 10 0.5000 Luc-LNP PBS S.C. 0.0200 501 0.0500 Luc-LNP PBS S.C. 0.0020 50 0.1 0.0050 Luc-LNP PBS S.C. 0.000250 0.01 0.0005 Luc-LNP PBS I.V. 0.2000 50 10 0.5000 Luc-LNP PBS I.V.0.0200 50 1 0.0500 Luc-LNP PBS I.V. 0.0020 50 0.1 0.0050 Luc-LNP PBSI.V. 0.0002 50 0.01 0.0005 Luc-LNP PBS I.M. 0.2000 50 10 0.5000 Luc-LNPPBS I.M. 0.0200 50 1 0.0500 Luc-LNP PBS I.M. 0.0020 50 0.1 0.0050Luc-LNP PBS I.M. 0.0002 50 0.01 0.0005 Luc-PBS PBS I.V. 0.20 50 10 0.50

The mice are imaged at 2, 8, 24, 48, 120 and 192 hours to determine thebioluminescence (measured as total flux (photons/second) of the entiremouse). At 8 hours or 192 hours the liver, spleen, kidney and injectionsite for subcutaneous and intramuscular administration are imaged todetermine the bioluminescence.

Example 90 Cationic Lipid Formulation Studies of Chemically ModifiedmRNA

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), pseudouridine (pU) orN1-methylpseudouridine (N1mpU) was formulated in the cationic lipidsdescribed in Table 137. The formulations were administered intravenously(I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C miceat a dose of 0.05 mg/kg.

TABLE 137 Cationic Lipid Formulations Formulation NPA-137-1 NPA-134-1NPA-135-1 NPA-136-1 111612-A Lipid DLin-MC3- DLin- DLin-KC2- C12-200DODMA DMA MC3-DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio (wt/wt)Mean Size 111 nm 104 nm 95 nm 143 nm 223.2 nm PDI: 0.15 PDI: 0.13 PDI:0.11 PDI: 0.12 PDI: 0.142 Zeta at pH 7.4 −4.1 mV −1.9 mV −1.0 mV 0.2 mV−3.09 mV Encaps. 97% 100% 100% 78% 64% (RiboGr) Chemistry pU N1mpU N1mpUN1mpU 5mC/pU

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 4.11E+05 p/s. The results of the imaging areshown in Table 138. Peak expression was seen for all three routes testedat 8 hours.

TABLE 138 Flux DLin-MC3- DLin-MC3- DLin-KC2- DMA DMA DMA C12-200 DODMA(pU) (N1mpU) (N1mpU) (N1mpU) (5mC/pU) Route Time Point Flux (p/s) Flux(p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 3.21E+08 1.24E+091.01E+09 9.00E+08 3.90E+07 I.V. 8 hrs 1.60E+09 3.22E+09 2.38E+091.11E+09 1.17E+07 I.V. 24 hrs  1.41E+08 3.68E+08 3.93E+08 8.06E+071.11E+07 I.M. 2 hrs 2.09E+07 3.29E+07 8.32E+07 9.43E+07 4.66E+06 I.M. 8hrs 2.16E+08 6.14E+08 1.00E+09 8.77E+07 7.05E+06 I.M. 24 hrs  1.23E+071.40E+08 5.09E+08 1.36E+07 1.14E+06 S.C. 2 hrs 2.32E+07 3.60E+072.14E+08 1.01E+08 3.11E+07 S.C. 8 hrs 5.55E+08 9.80E+08 4.93E+091.01E+09 8.04E+07 S.C. 24 hrs  1.81E+08 2.74E+08 2.12E+09 4.74E+071.34E+07

Example 91 Studies of Chemical Modified mRNA

Luciferase mRNA (SEQ ID NO: 16; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) fully modified withN4-acetylcytidine (N-4-acetyl), fully modified with 5-methoxyuridine(5meth), fully modified with N4-acetylcytidine andN1-methylpseudouridine (N4-acetyl/N1mpU) or fully modified with5-methylcytosine and 5-methoxyuridine (5mC/5-meth) was formulated inDLin-MC3-DMA as described in Table 139. The formulations wereadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 139 Cationic Lipid Formulations Formulation NPA-141-1 NPA-142-1NPA-143-1 NPA-144-1 Lipid DLin- DLin- DLin- DLin- MC3-DMA MC3-DMAMC3-DMA MC3-DMA Lipid/mRNA 20:1 20:1 20:1 20:1 ratio (wt/wt) Mean Size138 nm 116 nm 144 nm 131 nm PDI: 0.16 PDI: 0.15 PDI: 0.15 PDI: 0.15 Zetaat pH 7.4 −2.8 mV −2.8 mV −4.3 mV −5.0 mV Encaps. 97% 100% 75% 72%(RiboGr) Chemistry N4-acetyl 5meth N4-acetyl/ 5mC/5-meth N1mpU

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 6 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 2.70E+05 p/s. The results of the imaging areshown in Table 140.

TABLE 140 Flux N4-acetyl/ 5mC/5- N4-acetyl 5meth N1mpU meth Route TimePoint Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 9.17E+073.19E+06 4.21E+07 1.88E+06 I.V. 6 hrs 7.70E+08 9.28E+06 2.34E+087.75E+06 I.V. 24 hrs  6.84E+07 1.04E+06 3.55E+07 3.21E+06 I.M. 2 hrs8.59E+06 7.86E+05 5.30E+06 5.11E+05 I.M. 6 hrs 1.27E+08 8.88E+063.82E+07 3.17E+06 I.M. 24 hrs  4.46E+07 1.38E+06 2.00E+07 1.39E+06 S.C.2 hrs 1.83E+07 9.67E+05 4.45E+06 1.01E+06 S.C. 6 hrs 2.89E+08 1.78E+078.91E+07 1.29E+07 S.C. 24 hrs  6.09E+07 6.40E+06 2.08E+08 6.63E+06

Example 92 PLGA Microspheres

A. Synthesis of PLGA Microspheres

Polylacticglycolic acid (PLGA) microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA-ester cap (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75,50:50 LA:GA) or PLGA-acid cap (Lactel, Cat# B6013-2, inherent viscosity0.55-0.75, 50:50 LA:GA), polyvinylalcohol (PVA) (Sigma, Cat#348406-25G,MW 13-23 k) dichloromethane and water. Briefly, 0.4 ml of mRNA in water(W1) at 4 mg/ml was added to 2 ml of PLGA dissolved in dichloromethane(DCM) (O1) at concentrations ranging from 50-200 mg/ml of PLGA. TheW1/O1 emulsion was homogenized (IKA Ultra-Turrax Homogenizer, T18) for30 seconds at speed 4 (˜15,000 rpm). The W1/O1 emulsion was then addedto 250 ml of 1% PVA (W2) and homogenized for 1 minute at speed 5(˜19,000 rpm). Formulations were left to stir for 3 hours, then passedthrough a 100 μm nylon mesh strainer (Fisherbrand Cell Strainer, Cat#22-363-549) to remove larger aggregates, and finally washed bycentrifugation (10 min, 9,250 rpm, 4° C.). The supernatant was discardedand the PLGA pellets were resuspended in 5-10 ml of water, which wasrepeated 2×. The washed formulations were frozen in liquid nitrogen andthen lyophilized for 2-3 days.

B. Decreasing Homogenization Speed or PLGA Concentration

PLGA luciferase microspheres (luciferase mRNA shown in SEQ ID NO: 16;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcysotine and pseudouridine) weremade using the conditions described above with ester-capped PLGA. Afterwashing and resuspension with water, 100-200 μl of a PLGA microspheressample was used to measure particle size of the formulations by laserdiffraction (Malvern Mastersizer2000). The particle size of themicrospheres made by decreasing homogenization speed during the additionof the first emulsion to the second emulsion with a PLGA concentrationof 200 mg/ml is shown in Table 141 and the particle size of themicrospheres made by decreasing PLGA concentration in dichloromethane(DCM) is shown in Table 142 with a homogenization speed of 5 during theaddition of the first emulsion to the second emulsion.

TABLE 141 Decreasing Homogenization Speed Addition Homogenizer D50Average Speed Size (μm) 2 41.5 3 35.9 4 32.5 5 26.5 6 25.0

TABLE 142 Decreasing PLGA Concentration in DCM PLGA Concentration D50Average (mg/mL) Size (μm) 200 27.7 100 14.2 50 8.7

PLGA with an inherent viscosity of 0.55-0.75 either acid or ester-cappedwas used to make microspheres shown in Table 143. The particle size ofthe microspheres and the release kinetics were also determined and areshown in Table 143.

TABLE 143 Decreasing PLGA Concentration in DCM Theoretical Actual D50PLGA Addition mRNA mRNA Average concentration End- Homogenizer LoadingLoading Encap. Size Sample mg/ml group Speed (wt %) (wt %) Eff. % (μm) A200 Ester 3 0.4 0.14 45 38.7 B 200 Acid 3 0.4 0.06 18 31.3 C 200 Ester 50.4 0.13 41 32.2 D 200 Acid 5 0.4 0.07 22 28.0 E 100 Ester 5 0.8 0.15 2317.1 F 100 Acid 5 0.8 0.10 18 15.9

C. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Luciferase modified RNA (SEQ ID NO:16; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine)were deformulated and the integrity of the extracted modified RNA wasdetermined by automated electrophoresis (Bio-Rad Experion). Afterlyophilization, ˜10 mg of PLGA MS were weighed out in 2 ml eppendorftubes and deformulated by adding 1 ml of DCM and letting the samplesshake for 2-6 hrs. mRNA was extracted from the deformulated PLGAmicropsheres by adding 0.5 ml of water and shaking the sample overnight.Unformulated luciferase mRNA in water (Deform control) was spiked intoDCM and went through the deformulation process to be used as a control.The extracted modified mRNA was compared against unformulated modifiedmRNA and the deformulation control in order to test the integrity of theencapsulated modified mRNA. The majority of modified RNA was intact forbatch ID A, B, C, D, E, as compared to the deformulated control (Deformcontrol) and the unformulated control (Unform control).

D. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA micropsheres formulated with Luciferase modified RNA (SEQ ID NO:16; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine)were resuspended in TE buffer to a PLGA microsphere concentration of 80mg/ml in duplicate or triplicate. After resuspension, samples were keptincubating and shaking at 37° C. during the course of the study. Foreach time point (0.04, 0.25, 1.2, 4, and 7 days), the tubes werecentrifuged, the supernantant was removed and the pellet was resuspendedin 0.25 ml of fresh TE buffer. To determine the amount of modified RNAreleased from the PLGA microspheres, the modified RNA concentration inthe supernatant was determined by OD 260. The percent release, shown inTable 144, was calculated based on the total amount of modified RNA ineach sample. The release rate of mRNA formulations can be tailored byaltering the particle size, the PLGA concentration, and the acid versusester-end cap.

TABLE 144 Percent Release Batch A Batch C Batch E Time % Batch B % BatchD % Batch F (days) Release % Release Release % Release Release % Release0.04 7.1 13.6 9.5 9.5 21.2 21.7 0.25 18.0 23.3 17.5 24.0 31.3 37.7 1.226.3 29.1 22.8 31.6 41.0 46.5 4 33.5 37.1 29.0 40.4 48.8 60.7 7 37.641.5 32.4 45.2 55.0 68.3

E. Luciferase PLGA Microspheres in Vivo Study

PLGA microspheres containing luciferase mRNA (SEQ ID NO: 16; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1)fully modified with 5-methylcytosine and N1-methylpseudouridine or fullymodified with N1-methylpseudouridine are formulated as described inTable 145 and injected subcutaneously into mice. Twenty minutes prior toimaging, mice are injected intraperitoneally with a D-luciferin solutionat 150 mg/kg. Animals are then anesthetized and images were acquiredwith an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescencewas measured as total flux (photons/second) of the entire mouse.

TABLE 145 Formulation Group n Dose (ug) Volume (ul) Vehicle 1 6 50 2000.5% CMC, 5% Mannitol, 0.1% Polysorbate 80 2 6 50 200 5% Sucrose 3 3 5200 5% Sucrose

Example 93 Buffer Formulations

Modified mRNA may be formulated in water based buffers. Buffers whichare similar to biological systems are traditionally isotonic. Suchbuffers and buffer solutions may be prepared according to the followingguidelines. Example components are given in Table 146.

In some embodiments, calcium ions may be added to a buffer solution forformulations.

TABLE 146 Buffers Buffer Components Tris buffered saline Tris and sodiumchloride Phosphate buffered saline sodium chloride, sodium phosphate,and, in some formulations, potassium chloride and potassium phosphateRinger's lactate 130 mEq of sodium ion = 130 mmol/L (for one liter) 109mEq of chloride ion = 109 mmol/L 28 mEq of lactate = 28 mmol/L 4 mEq ofpotassium ion = 4 mmol/L 3 mEq of calcium ion = 1.5 mmol/L

Example 94 Lipid Nanoparticle Containing a Plurality of Modified mRNAs

EPO mRNA (SEQ ID NO: 9; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand N1-methylpseudouridine), G-CSF mRNA (SEQ ID NO: 6; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and N1-methylpseudouridine) and Factor IXmRNA (SEQ ID NO: 10; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand N1-methylpseudouridine), is formulated in DLin-MC3-DMA as describedin Table 147. The formulations are administered intravenously (I.V.),intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a doseof 0.05 mg/kg. Control LNP formulations containing only one mRNA arealso administered at an equivalent dose.

TABLE 147 DLin-MC3-DMA Formulation Formulation NPA-157-1 LipidDLin-MC3-DMA Lipid/mRNA 20:1 ratio (wt/wt) Mean Size 89 nm PDI: 0.08Zeta at pH 7.4 1.1 mV Encaps. 97% (RiboGr)

Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7days after administration of the formulation. The serum is analyzed byELISA to determine the protein expression of EPO, G-CSF, and Factor IX.

Example 95 Cationic Lipid Formulation Studies of 5-Methylcytosine andN1-Methylpseudouridine Modified mRNA

EPO mRNA (SEQ ID NO: 9; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand N1-methylpseudouridine) or G-CSF mRNA (SEQ ID NO: 6; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and N1-methylpseudouridine) is formulatedin DLin-MC3-DMA and DLin-KC2-DMA as described in Table 148. Theformulations are administered intravenously (I.V), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 148 DLin-MC3-DMA and DLin-KC2-DMA Formulations FormulationNPA-147-1 NPA-148-1 NPA-150-1 NPA-151-1 mRNA EPO EPO G-CSF G-CSF LipidDLin-MC3- DLin-KC2- DLin-MC3- DLin-KC2- DMA DMA DMA DMA Lipid/mRNA 20:120:1 20:1 20:1 ratio (wt/wt) Mean Size 117 nm 82 nm 119 nm 88 nm PDI:0.14 PDI: 0.08 PDI: 0.13 PDI: 0.08 Zeta at pH 7.4 −1.7 mV 0.6 mV 3.6 mV2.2 mV Encaps. 100% 96% 100% 100% (RiboGr)

Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7days after administration of the formulation. The serum is analyzed byELISA to determine the protein expression of EPO and G-CSF.

Example 96 Directed SAR of Pseudouridine and N1-Methyl PseudoUridine

With the recent focus on the pyrimidine nucleoside pseudouridine, aseries of structure-activity studies were designed to investigate mRNAcontaining modifications to pseudouridine or N1-methyl-pseudourdine.

The study was designed to explore the effect of chain length, increasedlipophilicity, presence of ring structures, and alteration ofhydrophobic or hydrophilic interactions when modifications were made atthe N1 position, C6 position, the 2-position, the 4-position and on thephosphate backbone. Stability is also investigated.

To this end, modifications involving alkylation, cycloalkylation,alkyl-cycloalkylation, arylation, alkyl-arylation, alkylation moietieswith amino groups, alkylation moieties with carboxylic acid groups, andalkylation moieties containing amino acid charged moieties areinvestigated. The degree of alkylation is generally C₁-C₆. Examples ofthe chemistry modifications include those listed in Table 149 and Table150.

TABLE 149 Pseudouridine and N1-methyl Pseudo Uridine SAR NaturallyChemistry Modification Compound # occuring N1-ModificationsN1-Ethyl-pseudo-UTP 1 N N1-Propyl-pseudo-UTP 2 NN1-iso-propyl-pseudo-UTP 3 N N1-(2,2,2-Trifluoroethyl)-pseudo-UTP 4 NN1-Cyclopropyl-pseudo-UTP 5 N N1-Cyclopropylmethyl-pseudo-UTP 6 NN1-Phenyl-pseudo-UTP 7 N N1-Benzyl-pseudo-UTP 8 NN1-Aminomethyl-pseudo-UTP 9 N Pseudo-UTP-N1-2-ethanoic acid 10 NN1-(3-Amino-3-carboxypropyl)pseudo-UTP 11 NN1-Methyl-3-(3-amino-3-carboxypropyl)- 12 Y pseudo-UTP C-6 Modifications6-Methyl-pseudo-UTP 13 N 6-Trifluoromethyl-pseudo-UTP 14 N6-Methoxy-pseudo-UTP 15 N 6-Phenyl-pseudo-UTP 16 N 6-Iodo-pseudo-UTP 17N 6-Bromo-pseudo-UTP 18 N 6-Chloro-pseudo-UTP 19 N 6-Fluoro-pseudo-UTP20 N 2- or 4-position Modifications 4-Thio-pseudo-UTP 21 N2-Thio-pseudo-UTP 22 N Phosphate backbone ModificationsAlpha-thio-pseudo-UTP 23 N N1-Me-alpha-thio-pseudo-UTP 24 N

TABLE 150 Pseudouridine and N1-methyl Pseudo Uridine SAR NaturallyChemistry Modification Compound # occuring N1-Methyl-pseudo-UTP  1 YN1-Butyl-pseudo-UTP  2 N N1-tert-Butyl-pseudo-UTP  3 NN1-Pentyl-pseudo-UTP  4 N N1-Hexyl-pseudo-UTP  5 NN1-Trifluoromethyl-pseudo-UTP  6 Y N1-Cyclobutyl-pseudo-UTP  7 NN1-Cyclopentyl-pseudo-UTP  8 N N1-Cyclohexyl-pseudo-UTP  9 NN1-Cycloheptyl-pseudo-UTP 10 N N1-Cyclooctyl-pseudo-UTP 11 NN1-Cyclobutylmethyl-pseudo-UTP 12 N N1-Cyclopentylmethyl-pseudo-UTP 13 NN1-Cyclohexylmethyl-pseudo-UTP 14 N N1-Cycloheptylmethyl-pseudo-UTP 15 NN1-Cyclooctylmethyl-pseudo-UTP 16 N N1-p-tolyl-pseudo-UTP 17 NN1-(2,4,6-Trimethyl-phenyl)pseudo-UTP 18 NN1-(4-Methoxy-phenyl)pseudo-UTP 19 N N1-(4-Amino-phenyl)pseudo-UTP 20 NN1(4-Nitro-phenyl)pseudo-UTP 21 N Pseudo-UTP-N1-p-benzoic acid 22 NN1-(4-Methyl-benzyl)pseudo-UTP 24 NN1-(2,4,6-Trimethyl-benzyl)pseudo-UTP 23 NN1-(4-Methoxy-benzyl)pseudo-UTP 25 N N1-(4-Amino-benzyl)pseudo-UTP 26 NN1-(4-Nitro-benzyl)pseudo-UTP 27 N Pseudo-UTP-N1-methyl-p-benzoic acid28 N N1-(2-Amino-ethyl)pseudo-UTP 29 N N1-(3-Amino-propyl)pseudo-UTP 30N N1-(4-Amino-butyl)pseudo-UTP 31 N N1-(5-Amino-pentyl)pseudo-UTP 32 NN1-(6-Amino-hexyl)pseudo-UTP 33 N Pseudo-UTP-N1-3-propionic acid 34 NPseudo-UTP-N1-4-butanoic acid 35 N Pseudo-UTP-N1-5-pentanoic acid 36 NPseudo-UTP-N1-6-hexanoic acid 37 N Pseudo-UTP-N1-7-heptanoic acid 38 NN1-(2-Amino-2-carboxyethyl)pseudo-UTP 39 NN1-(4-Amino-4-carboxybutyl)pseudo-UTP 40 N N3-Alkyl-pseudo-UTP 41 N6-Ethyl-pseudo-UTP 42 N 6-Propyl-pseudo-UTP 43 N 6-iso-Propyl-pseudo-UTP44 N 6-Butyl-pseudo-UTP 45 N 6-tert-Butyl-pseudo-UTP 46 N6-(2,2,2-Trifluoroethyl)-pseudo-UTP 47 N 6-Ethoxy-pseudo-UTP 48 N6-Trifluoromethoxy-pseudo-UTP 49 N 6-Phenyl-pseudo-UTP 50 N6-(Substituted-Phenyl)-pseudo-UTP 51 N 6-Cyano-pseudo-UTP 52 N6-Azido-pseudo-UTP 53 N 6-Amino-pseudo-UTP 54 N6-Ethylcarboxylate-pseudo-UTP  54b N 6-Hydroxy-pseudo-UTP 55 N6-Methylamino-pseudo-UTP  55b N 6-Dimethylamino-pseudo-UTP 57 N6-Hydroxyamino-pseudo-UTP 59 N 6-Formyl-pseudo-UTP 60 N6-(4-Morpholino)-pseudo-UTP 61 N 6-(4-Thiomorpholino)-pseudo-UTP 62 NN1-Me-4-thio-pseudo-UTP 63 N N1-Me-2-thio-pseudo-UTP 64 N1,6-Dimethyl-pseudo-UTP 65 N 1-Methyl-6-trifluoromethyl-pseudo-UTP 66 N1-Methyl-6-ethyl-pseudo-UTP 67 N 1-Methyl-6-propyl-pseudo-UTP 68 N1-Methyl-6-iso-propyl-pseudo-UTP 69 N 1-Methyl-6-butyl-pseudo-UTP 70 N1-Methyl-6-tert-butyl-pseudo-UTP 71 N1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP 72 N1-Methyl-6-iodo-pseudo-UTP 73 N 1-Methyl-6-bromo-pseudo-UTP 74 N1-Methyl-6-chloro-pseudo-UTP 75 N 1-Methyl-6-fluoro-pseudo-UTP 76 N1-Methyl-6-methoxy-pseudo-UTP 77 N 1-Methyl-6-ethoxy-pseudo-UTP 78 N1-Methyl-6-trifluoromethoxy-pseudo-UTP 79 N 1-Methyl-6-phenyl-pseudo-UTP80 N 1-Methyl-6-(substituted phenyl)pseudo-UTP 81 N1-Methyl-6-cyano-pseudo-UTP 82 N 1-Methyl-6-azido-pseudo-UTP 83 N1-Methyl-6-amino-pseudo-UTP 84 N 1-Methyl-6-ethylcarboxylate-pseudo-UTP85 N 1-Methyl-6-hydroxy-pseudo-UTP 86 N1-Methyl-6-methylamino-pseudo-UTP 87 N1-Methyl-6-dimethylamino-pseudo-UTP 88 N1-Methyl-6-hydroxyamino-pseudo-UTP 89 N 1-Methyl-6-formyl-pseudo-UTP 90N 1-Methyl-6-(4-morpholino)-pseudo-UTP 91 N1-Methyl-6-(4-thiomorpholino)-pseudo-UTP 92 N 1-Alkyl-6-vinyl-pseudo-UTP93 N 1-Alkyl-6-allyl-pseudo-UTP 94 N 1-Alkyl-6-homoallyl-pseudo-UTP 95 N1-Alkyl-6-ethynyl-pseudo-UTP 96 N 1-Alkyl-6-(2-propynyl)-pseudo-UTP 97 N1-Alkyl-6-(1-propynyl)-pseudo-UTP 98 N

Example 97 Incorporation of Naturally and Non-Naturally OccurringNucleosides

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Examples of these are given inTables 151 and 152. Certain commercially available nucleosidetriphosphates (NTPs) are investigated in the polynucleotides of theinvention. A selection of these are given in Table 152. The resultantmRNA are then examined for their ability to produce protein, inducecytokines, and/or produce a therapeutic outcome.

TABLE 151 Naturally and non-naturally occurring nucleosides NaturallyChemistry Modification Compound # occuring N4-Methyl-Cytosine 1 YN4,N4-Dimethyl-2′-OMe-Cytosine 2 Y 5-Oxyacetic acid-methyl ester-Uridine3 Y N3-Methyl-pseudo-Uridine 4 Y 5-Hydroxymethyl-Cytosine 5 Y5-Trifluoromethyl-Cytosine 6 N 5-Trifluoromethyl-Uridine 7 N5-Methyl-amino-methyl-Uridine 8 Y 5-Carboxy-methyl-amino-methyl-Uridine9 Y 5-Carboxymethylaminomethyl-2′-OMe-Uridine 10 Y5-Carboxymethylaminomethyl-2-thio-Uridine 11 Y5-Methylaminomethyl-2-thio-Uridine 12 Y5-Methoxy-carbonyl-methyl-Uridine 13 Y5-Methoxy-carbonyl-methyl-2′-OMe-Uridine 14 Y 5-Oxyacetic acid-Uridine15 Y 3-(3-Amino-3-carboxypropyl)-Uridine 16 Y5-(carboxyhydroxymethyl)uridine methyl ester 17 Y5-(carboxyhydroxymethyl)uridine 18 Y

TABLE 152 Non-naturally occurring nucleoside triphosphates NaturallyChemistry Modification Compound # occuring N1-Me-GTP 1 N2′-OMe-2-Amino-ATP 2 N 2′-OMe-pseudo-UTP 3 Y 2′-OMe-6-Me-UTP 4 N2′-Azido-2′-deoxy-ATP 5 N 2′-Azido-2′-deoxy-GTP 6 N2′-Azido-2′-deoxy-UTP 7 N 2′-Azido-2′-deoxy-CTP 8 N2′-Amino-2′-deoxy-ATP 9 N 2′-Amino-2′-deoxy-GTP 10 N2′-Amino-2′-deoxy-UTP 11 N 2′-Amino-2′-deoxy-CTP 12 N 2-Amino-ATP 13 N8-Aza-ATP 14 N Xanthosine-5′-TP 15 N 5-Bromo-CTP 16 N2′-F-5-Methyl-2′-deoxy-UTP 17 N 5-Aminoallyl-CTP 18 N2-Amino-riboside-TP 19 N

Example 98 Incorporation of Modifications to the Nucleobase andCarbohydrate (Sugar)

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Commercially availablenucleosides and NTPs having modifications to both the nucleobase andcarbohydrate (sugar) are examined for their ability to be incorporatedinto mRNA and to produce protein, induce cytokines, and/or produce atherapeutic outcome. Examples of these nucleosides are given in Tables153 and 154.

TABLE 153 Combination modifications Chemistry Modification Compound #5-iodo-2′-fluoro-deoxyuridine 1 5-iodo-cytidine 6 2′-bromo-deoxyuridine7 8-bromo-adenosine 8 8-bromo-guanosine 9 2,2′-anhydro-cytidinehydrochloride 10 2,2′-anhydro-uridine 11 2′-Azido-deoxyuridine 122-amino-adenosine 13 N4-Benzoyl-cytidine 14 N4-Amino-cytidine 152′-O-Methyl-N4-Acetyl-cytidine 16 2′Fluoro-N4-Acetyl-cytidine 172′Fluor-N4-Bz-cytidine 18 2′O-methyl-N4-Bz-cytidine 192′O-methyl-N6-Bz-deoxyadenosine 20 2′Fluoro-N6-Bz-deoxyadenosine 21N2-isobutyl-guanosine 22 2′Fluro-N2-isobutyl-guanosine 232′O-methyl-N2-isobutyl-guanosine 24

TABLE 154 Naturally occuring combinations Naturally Name Compound #occurring 5-Methoxycarbonylmethyl-2-thiouridine TP 1 Y5-Methylaminomethyl-2-thiouridine TP 2 Y 5-Crbamoylmethyluridine TP 3 Y5-Carbamoylmethyl-2′-O-methyluridine TP 4 Y1-Methyl-3-(3-amino-3-carboxypropyl) 5 Y pseudouridine TP5-Methylaminomethyl-2-selenouridine TP 6 Y 5-Carboxymethyluridine TP 7 Y5-Methyldihydrouridine TP 8 Y lysidine TP 9 Y 5-Taurinomethyluridine TP10 Y 5-Taurinomethyl-2-thiouridine TP 11 Y5-(iso-Pentenylaminomethyl)uridine TP 12 Y5-(iso-Pentenylaminomethyl)-2-thiouridine TP 13 Y5-(iso-Pentenylaminomethyl)-2′-O- 14 Y methyluridine TPN4-Acetyl-2′-O-methylcytidine TP 15 Y N4,2′-O-Dimethylcytidine TP 16 Y5-Formyl-2′-O-methylcytidine TP 17 Y 2′-O-Methylpseudouridine TP 18 Y2-Thio-2′-O-methyluridine TP 19 Y 3,2′-O-Dimethyluridine TP 20 Y

In the tables “UTP” stands for uridine triphosphate, “GTP” stands forguanosine triphosphate, “ATP” stands for adenosine triphosphate, “CTP”stands for cytosine triphosphate, “TP” stands for triphosphate and “Bz”stands for benzyl.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

What is claimed is:
 1. A method for producing a protein of interest in amammal comprising contacting said mammal with a modified mRNA comprisingSEQ ID NO: 10 formulated in a lipid nanoparticle.
 2. The method of claim1 wherein the lipid nanoparticle has a final total molar ratio of 50 mol% cationic lipid, 10 mol % DSPC, 38.5 mol % Cholesterol and 1.5 mol % ofPEG lipid.
 3. The method of claim 2, wherein the lipid nanoparticle hasa 20:1 weight ratio of total lipid to the modified RNA.
 4. The method ofclaim 2, wherein the cationic lipid is DLin-KC2-DMA.
 5. The method ofclaim 4, wherein the PEG lipid is PEG-c-DOMG.
 6. The method of claim 5,and wherein said protein of interest is detectable 8 hours aftercontacting said mammal.
 7. The method of claim 1, wherein the modifiedmRNA comprises at least one nucleoside modification.